MN gene and protein

ABSTRACT

A new gene—MN—and proteins/polypeptides encoded therefrom are disclosed. Recombinant nucleic acid molecules for expressing MN proteins/polypeptides and recombinant proteins are provided. Expression of the MN gene is disclosed as being associated with tumorigenicity, and the invention concerns methods and compositions for detecting and/or quantitating MN antigen and/or MN-specific antibodies in vertebrate samples that are diagnostic/prognostic for neoplastic and pre-neoplastic disease. Test kits embodying the immunoassays of this invention are provided. MN-specific antibodies are disclosed that can be used diagnostically/prognostically, therapeutically, for imaging, and/or for affinity purification of MN proteins/polypeptides. Also provided are nucleic acid probes for the MN gene as well as test kits comprising said probes. The invention also concerns vaccines comprising MN proteins/polypeptides which are effective to immunize a vertebrate against neoplastic diseases associated with the expression of MN proteins. The invention still further concerns antisense nucleic acid sequences that can be used to inhibit MN gene expression.

[0001] This application is a continuation-in-part of now pending U.S.Ser. No. 08/177,093 (filed Dec. 30, 1993), which is in turn acontinuation-in-part of U.S. Ser. No. 07/964,589 (filed Oct. 21, 1992).This application declares priority under 35 USC § 120 from those U.S.applications, and also under 35 USC § 119 from the now pendingCzechoslovakian patent application PV-709-92 (filed Mar. 11, 1992).

FIELD OF THE INVENTION

[0002] The present invention is in the general area of medical geneticsand in the fields of biochemical engineering and immunochemistry. Morespecifically, it relates to the identification of a new gene—the MNgene—a cellular gene coding for the MN protein. The inventors hereoffound MN proteins to be associated with tumorigenicity. Evidenceindicates that the MN protein appears to represent a potentially noveltype of oncoprotein. Identification of MN antigen as well as antibodiesspecific therefor in patient samples provides the basis fordiagnostic/prognostic assays for cancer.

BACKGROUND OF THE INVENTION

[0003] A novel quasi-viral agent having rather unusual properties wasdetected by its capacity to complement mutants of vesicular stomatitisvirus (VSV) with heat-labile surface G protein in HeLa cells (cell linederived from human cervical adenocarcinoma), which had been cocultivatedwith human breast carcinoma cells. [Zavada et al., Nature New Biol. 240:124 (1972); Zavada et al., J. Gen. Virol., 24: 327 (1974); Zavada, J.Arch. Virol., 50: 1 (1976); Zavada, J., J. Gen. Virol., 63: 15-24(1982); Zavada and Zavadova, Arch. Virol., 118: 189 (1991).] The quasiviral agent was called MaTu as it was presumably derived from a humanmammary tumor.

[0004] There was significant medical interest in studying andcharacterizing MaTu as it appeared to be an entirely new type ofmolecular parasite of living cells, and possibly originated from a humantumor. Described herein is the elucidation of the biological andmolecular nature of MaTu which resulted in the discovery of the MN geneand protein. MaTu was found by the inventors to be a two-componentsystem, having an exogenous transmissible component, MX, and anendogenous cellular component, MN. As described herein, the MN componentwas found to be a cellular gene, showing only very little homology withknown DNA sequences. The MN gene was found to be present in thechromosomal DNA of all vertebrates tested, and its expression was foundto be strongly correlated with tumorigenicity.

[0005] The exogenous MaTu-MX transmissible agent was identified aslymphocytic choriomeningitis virus (LCMV) which persistently infectsHeLa cells. The inventors discovered that the MN expression in HeLacells is positively regulated by cell density, and also its expressionlevel is increased by persistent infection with LCMV.

[0006] Research results provided herein show that cells transfected withMN cDNA undergo changes indicative of malignant transformation. Furtherresearch findings described herein indicate that the disruption of cellcycle control is one of the mechanisms by which MN may contribute to thecomplex process of tumor development.

[0007] Described herein is the cloning and sequencing of the MN gene andthe recombinant production of MN proteins. Also described are antibodiesprepared against MN proteins/polypeptides. MN proteins/polypeptides canbe used in serological assays according to this invention to detectMN-specific antibodies. Further, MN proteins/polypeptides and/orantibodies reactive with MN antigen can be used in immunoassaysaccording to this invention to detect and/or quantitate MN antigen. Suchassays may be diagnostic and/or prognostic for neoplastic/pre-neoplasticdisease.

SUMMARY OF THE INVENTION

[0008] Herein disclosed is the MN gene, a cellular gene which is theendogenous component of the MaTu agent. cDNA sequences for the MN geneare shown in FIGS. 1A-B [SEQ. ID. NO.: 1] and FIG. 15 [SEQ. ID. NO.: 5].FIG. 25 provides the sequence of a MN genomic clone containing apromoter region [SEQ. ID. NO.: 23].

[0009] This invention is directed to said MN gene, fragments thereof andthe related cDNA which are useful, for example, as follows: 1) toproduce MN proteins/polypeptides by biochemical engineering; 2) toprepare nucleic acid probes to test for the presence of the MN gene incells of a subject; 3) to prepare appropriate polymerase chain reaction(PCR) primers for use, for example, in PCR-based assays or to producenucleic acid probes; 4) to identify MN proteins and polypeptides as wellas homologs or near homologs thereto; 5) to identify various mRNAstranscribed from MN genes in various tissues and cell lines, preferablyhuman; and 6) to identify mutations in MN genes. The invention furtherconcerns purified and isolated DNA molecules comprising the MN gene orfragments thereof, or the related cDNA or fragments thereof.

[0010] Thus, this invention in one aspect concerns isolated nucleic acidsequences that encode MN proteins or polypeptides wherein the nucleotidesequences for said nucleic acids are selected from the group consistingof:

[0011] (a) SEQ. ID. NO.: 1;

[0012] (b) SEQ. ID. NO.: 5;

[0013] (c) nucleotide sequences that hybridize under stringentconditions to SEQ. ID. NO.: 1 or to its complement;

[0014] (d) nucleotide sequences that hybridize under stringentconditions to SEQ. ID. NO.: 5 or to its complement; and

[0015] (e) nucleotide sequences that differ from SEQ. ID. NO.: 1 or SEQ.ID NO.: 5, or from the nucleotide sequences of (c) and (d) in codonsequence because of the degeneracy of the genetic code, that is,sequences that are degenerate variants of those sequences. Further, suchnucleic acid sequences are selected from nucleotide sequences that butfor the degeneracy of the genetic code would hybridize to either SEQ.ID. NO.: 1 or SEQ. ID. NO.: 5 under stringent hybridization conditions.

[0016] Further, such isolated nucleic acids that encode MN proteins orpolypeptides can also include the MN nucleic acid of the genomic cloneshown in FIG. 25a-b, that is, SEQ. ID. NO.: 23, as well as sequencesthat hybridize to it or its complement under stringent conditions, orwould hybridize to SEQ. ID. NO.: 23 or its complement under suchconditions, but for the degeneracy of the genetic code.

[0017] Further, this invention concerns nucleic acid probes which arefragments of the isolated nucleic acids that encode MN proteins orpolypeptides as described above. Preferably said nucleic acid probes arecomprised of at least 50 nucleotides, more preferably at least 100nucleotides, and still more preferably at least 150 nucleotides.

[0018] Still further, this invention is directed to isolated nucleicacids selected from the group consisting of:

[0019] (a) a nucleic acid having the nucleotide sequence shown in FIG.25 [SEQ. ID. NO.: 23] and its complement;

[0020] (b) nucleic acids that hybridize under standard stringenthybridization conditions to the nucleic acid of

[0021] (a) or to its complement; and

[0022] (c) nucleic acids that differ from the nucleic acids of (a) and(b) in codon sequence because of the degeneracy of the genetic code. Theinvention also concerns nucleic acids that but for the degeneracy of thegenetic code would hybridize to the nucleic acid of (a) or to itscomplement under standard stringent hybridization conditions. Thenucleic acids of (b) and (c) that hybridize to the coding region of SEQ.ID. NO.: 23 preferably have a length of at least 50 nucleotides, whereasthe nucleic acids of (b) and (c) that hybridize partially or wholly tothe non-coding region of SEQ. ID. NO.: 23 or its complement are thosethat function as nucleic acid probes to identify MN nucleic acidsequences. Conventional technology can be used to determine whether thenucleic acids of (b) and (c) or of fragments of SEQ. ID. NO.: 23 areuseful to identify MN nucleic acid sequences, for example, as outlinedin Benton and Davis, Science. 196: 180 (1977) and Fuscoe et al.Genomics. 5: 100 (1989). In general, the nucleic acids of (b) and (c)are preferably at least 50 nucleotides, more preferably at least 100nucleotides, and still more preferably at least 150 nucleotides.

[0023] Test kits of this invention can comprise the nucleic acid probesof the invention which are useful diagnostically/prognostically forneoplastic and/or pre-neoplastic disease. Preferred test kits comprisemeans for detecting or measuring the hybridization of said probes to theMN gene or to the mRNA product of the MN gene, such as a visualizingmeans.

[0024] Fragments of the isolated nucleic acids of the invention, canalso be used as PCR primers to amplify segments of MN genes, and may beuseful in identifying mutations in MN genes. Typically, said PCR primersare olignucleotides, preferably at least 16 nucleotides, but they may beconsiderably longer. Exemplary primers may be from about 16 nucleotidesto about 50 nucleotides, preferably from about 19 nucleotides to about45 nucleotides.

[0025] This invention also concerns nucleic acids which encode MNproteins or polypeptides that are specifically bound by monoclonalantibodies designated M75 that are produced by the hybridoma VU-M75deposited at the American Type Culture Collection (ATCC) at 12301Parklawn Drive in Rockville, Md. 20852 (USA) under ATCC No. HB 11128,and/or by monoclonal antibodies designated MN12 produced by thehybridoma MN 12.2.2 deposited at the ATCC under ATCC No. HB 11647.

[0026] The invention further concerns the discovery of a hithertounknown protein—MN, encoded by the MN gene. The expresssion of MNproteins is inducible by growing cells in dense cultures, and suchexpression was discovered to be associated with tumorigenic cells.

[0027] MN proteins were found to be produced by some human tumor celllines in vitro, for example, by HeLa (cervical carcinoma), T24 (bladdercarcinoma) and T47D (mammary carcinoma) and SK-Mel 1477 (melanoma) celllines, by tumorigenic hybrid cells and by cells of some human cancers invivo, for example, by cells of uterine cervical, ovarian and endometrialcarcinomas as well as cells of some benign neoplasias such as mammarypapillomas. MN proteins were not found in non-tumorigenic hybrid cells,and are generally not found in the cells of normal tissues, althoughthey have been found in a few normal tissues, most notably andabundantly in normal stomach tissues. MN antigen was found byimmunohistochemical staining to be prevalent in tumor cells and to bepresent sometimes in morphologically normal appearing areas of tissuespecimens exhibiting dysplasia and/or malignancy. Thus, the MN gene isstrongly correlated with tumorigenesis and is considered to be aputative oncogene.

[0028] In HeLa and in tumorigenic HeLa x fibroblast hybrid (H/F-T)cells, MN protein is manifested as a “twin” protein p54/58N; it isglycosylated and forms disulfide-linked oligomers. As determined byelectrophoresis upon reducing gels, MN proteins have molecular weightsin the range of from about 40 kd to about 70 kd, preferably from about45 kd to about 65 kd, more preferably from about 48 kd to about 58 kd.Upon non-reducing gels, MN proteins in the form of oligomers havemolecular weights in the range of from about 145 kd to about 160 kd,preferably from about 150 to about 155 kd, still more preferably fromabout 152 to about 154 kd. The predicted amino acid sequences forpreferred MN proteins of this invention are shown in FIG. 1A-1B [SEQ.ID. NO. 2] and in FIG. 15 [SEQ. ID. NO.: 6].

[0029] The discovery of the MN gene and protein and thus, ofsubstantially complementary MN genes and proteins encoded thereby, ledto the finding that the expression of MN proteins was associated withtumorigenicity. That finding resulted in the creation of methods thatare diagnostic/prognostic for cancer and precancerous conditions.Methods and compositions are provided for identifying the onset andpresence of neoplastic disease by detecting and/or quantitating MNantigen in patient samples, including tissue sections and smears, celland tissue extracts from vertebrates, preferably mammals and morepreferably humans. Such MN antigen may also be found in body fluids.

[0030] MN proteins and genes are of use in research concerning themolecular mechanisms of oncogenesis, in cancer diagnostics/prognostics,and may be of use in cancer immunotherapy. The present invention isuseful for detecting a wide variety of neoplastic and/or pre-neoplasticdiseases. Exemplary neoplastic diseases include carcinomas, such asmammary, bladder, ovarian, uterine, cervical, endometrial, squamous celland adenosquamous carcinomas; and head and neck cancers; mesodermaltumors, such as neuroblastomas and retinoblastomas; sarcomas, such asosteosarcomas and Ewing's sarcoma; and melanomas. Of particular interestare head and neck cancers, gynecologic cancers including ovarian,cervical, vaginal, endometrial and vulval cancers; gastrointestinalcancer, such as, stomach, colon and esophageal cancers; urinary tractcancer, such as, bladder and kidney cancers; skin cancer; liver cancer;prostate cancer; lung cancer; and breast cancer. Of still furtherparticular interest are gynecologic cancers; breast cancer; urinarytract cancers, especially bladder cancer; lung cancer; and liver cancer.Even further of particular interest are gynecologic cancers and breastcancer. Gynecologic cancers of particular interest are carcinomas of theuterine cervix, endometrium and ovaries; more particularly suchgynecologic cancers include cervical squamous cell carcinomas,adenosquamous carcinomas, adenocarcinomas as well as gynecologicprecancerous conditions, such as metaplastic cervical tissues andcondylomas.

[0031] The invention further relates to the biochemical engineering ofthe MN gene, fragments thereof or related cDNA. For example, said geneor a fragment thereof or related cDNA can be inserted into a suitableexpression vector; host cells can be transformed with such an expressionvector; and an MN protein/polypeptide, preferably an MN protein, isexpressed therein. Such a recombinant protein or polypeptide can beglycosylated or nonglycosylated, preferably glycosylated, and can bepurified to substantial purity. The invention further concerns MNproteins/polypeptides which are synthetically or otherwise biologicallyprepared.

[0032] Said MN proteins/polypeptides can be used in assays to detect MNantigen in patient samples and in serological assays to test forMN-specific antibodies. MN proteins/polypeptides of this invention areserologically active, immunogenic and/or antigenic. They can further beused as immunogens to produce MN-specific antibodies, polyclonal and/ormonoclonal, as well as an immune T-cell response.

[0033] The invention further is directed to MN-specific antibodies,which can be used diagnostically/prognostically and may be usedtherapeutically. Preferred according to this invention are MN-specificantibodies reactive with the epitopes represented respectively by theamino acid sequences of the MN protein shown in FIG. 15 as follows: fromAA 62 to AA 67 [SEQ. ID. NO.: 10]; from AA 55 to AA 60 [SEQ. ID. NO.:11]; from AA 127 to AA 147 [SEQ. ID. NO.: 12]; from AA 36 to AA 51 [SEQ.ID. NO.: 13]; from AA 69 to AA 83 [SEQ. ID. NO.: 14]; from AA 279 to AA291 [SEQ. ID. NO.: 15]; and from AA 450 to AA 462 [SEQ. ID. NO.: 16].More preferred are antibodies reactive with epitopes represented by SEQ.ID. NOS.: 10, 11 and 12. Still more preferred are antibodies reactivewith the epitopes represented by SEQ. ID NOS: 10 and 11, as for example,respectively Mabs M75 and MN12. Most preferred are monoclonal antibodiesreactive with the epitope represented by SEQ. ID. NO.: 10.

[0034] Also preferred according to this invention are antibodiesprepared against recombinantly produced MN proteins as, for example,GEX-3X-MN and MN 20-19. Also preferred are MN-specific antibodiesprepared against glycosylated MN proteins, such as, MN 20-19 expressedin baculovirus infected Sf9 cells.

[0035] A hybridoma that produces a representative MN-specific antibody,the monoclonal antibody M75 (Mab M75), was deposited at the under ATCCNumber HB 11128 as indicated above. The M75 antibody was used todiscover and identify the MN protein and can be used to identify readilyMN antigen in Western blots, in radioimmunoassays andimmunohistochemically, for example, in tissue samples that are fresh,frozen, or formalin-, alcohol-, acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Another representative MN-specificantibody, Mab MN12, is secreted by the hybridoma MN 12.2.2, which wasdeposited at the ATCC under the designation HB 11647.

[0036] MN-specific antibodies can be used, for example, in laboratorydiagnostics, using immunofluorescence microscopy or immunohistochemicalstaining; as a component in immunoassays for detecting and/orquantitating MN antigen in, for example, clinical samples; as probes forimmunoblotting to detect MN antigen; in immunoelectron microscopy withcolloid gold beads for localization of MN proteins and/or polypeptidesin cells; and in genetic engineering for cloning the MN gene orfragments thereof, or related cDNA. Such MN-specific antibodies can beused as components of diagnostic/prognostic kits, for example, for invitro use on histological sections; such antibodies can also and usedfor in vivo diagnostics/prognostics, for example, such antibodies can belabeled appropriately, as with a suitable radioactive isotope, and usedin vivo to locate metastases by scintigraphy. Further such antibodiesmay be used in vivo therapeutically to treat cancer patients with orwithout toxic and/or cytostatic agents attached thereto. Further, suchantibodies can be used in vivo to detect the presence of neoplasticand/or pre-neoplastic disease. Still further, such antibodies can beused to affinity purify MN proteins and polypeptides.

[0037] This invention also concerns recombinant DNA molecules comprisinga DNA sequence that encodes for an MN protein or polypeptide, and alsorecombinant DNA molecules that encode not only for an MN protein orpolypeptide but also for an amino acid sequence of a non-MN protein orpolypeptide. Said non-MN protein or polypeptide may preferably benonimmunogenic to humans and not typically reactive to antibodies inhuman body fluids. Examples of such a DNA sequence is the alpha-peptidecoding region of beta-galactosidase and a sequence coding forglutathione S-transferase or a fragment thereof. However, in someinstances, a non-MN protein or polypeptide that is serologically active,immunogenic and/or antigenic may be preferred as a fusion partner to aMN antigen. Further, claimed herein are such recombinant fusionproteins/polypeptides which are substantially pure and non-naturallyoccurring. An exemplary fusion protein of this invention is GEX-3X-MN.

[0038] This invention also concerns methods of treating neoplasticdisease and/or pre-neoplastic disease comprising inhibiting theexpression of MN genes by administering antisense nucleic acid sequencesthat are substantially complementary to mRNA transcribed from MN genes.Said antisense nucleic acid sequences are those that hybridize to suchmRNA under stringent hybridization conditions. Preferred are antisensenucleic acid sequences that are substantially complementary to sequencesat the 5′ end of the MN cDNA sequences shown in FIG. 1A-1B and/or inFIG. 15. Preferably said antisense nucleic acid sequences areoligonucleotides.

[0039] This invention also concerns vaccines comprising an immunogenicamount of one or more substantially pure MN proteins and/or polypeptidesdispersed in a physiologically acceptable, nontoxic vehicle, whichamount is effective to immunize a vertebrate, preferably a mammal, morepreferably a human, against a neoplastic disease associated with theexpression of MN proteins. Said proteins can be recombinantly,synthetically or otherwise biologically produced. Recombinent MNproteins include GEX-3X-MN and MN 20-19. A particular use of saidvaccine would be to prevent recidivism and/or metastasis. For example,it could be administered to a patient who has had an MN-carrying tumorsurgically removed, to prevent recurrence of the tumor.

[0040] The immunoassays of this invention can be embodied in test kitswhich comprise MN proteins/polypeptides and/or MN-specific antibodies.Such test kits can be in solid phase formats, but are not limitedthereto, and can also be in liquid phase format, and can be based onimmunohistochemical assays, ELISAS, particle assays, radiometric orfluorometric assays either unamplified or amplified, using, for example,avidin/biotin technology. Abbreviations The following abbreviations areused herein.: AA amino acid ATCC American Type Culture Collection bpbase pairs BSA bovine serum albumin BRL Bethesda Research LaboratoriesCA carbonic anhydrase Ci curie cm centimeter cpm counts per minuteC-terminus carboxyl-terminus ° C. degrees centigrade DABdiaminobenzidine dH₂0 deionized water DMEM Dulbecco modified Eaglemedium DTT dithiothreitol EDTA ethylenediaminetetracetate EIA enzymeimmunoassay ELISA enzyme-linked immunosorbent assay EtOH ethanol Ffibroblasts FCS fetal calf serum FIBR fibroblasts FITC fluoresceinisothiocyanate GEX-3X-MN fusion protein MN glutathione S-transferase HHeLa cells H₂0₂ hydrogen peroxide HCA Hydrophobic Cluster Analysis HEFhuman embryo fibroblasts HeLa K standard type of HeLa cells HeLa SStanbridge's mutant HeLa D98/AH.2 H/F-T hybrid HeLa fibroblast cellsthat are tumorigenic; derived from HeLa D98/AH.2 H/F-N hybrid HeLafibroblast cells that are nontumorigenic; derived from HeLa D98/AH.2HGPRT⁻ hypoxanthine guanine phosphoribosyl transferase-deficient HLHhelix-loop-helix HRP horseradish peroxidase IPTGisopropyl-Beta-D-thiogalacto-pyranoside kb kilobase kbp kilobase pairskd kilodaltons KPL Kirkegaard & Perry Laboratories, Inc. LCMVlymphocytic choriomeningitis virus LTR long terminal repeat M molar mAmilliampere MAb monoclonal antibody ME mercaptoethanol MEM minimalessential medium min. minute(s) mg milligram ml milliliter mM millimolarMMC mitomycin C MTV mammary tumor virus N normal concentration ngnanogram NGS normal goat serum N-terminus amino-terminus ODNoligodeoxynucleotide PAGE polyacrylamide gel electrophoresis PBSphosphate buffered saline PCR polymerase chain reaction PEST combinationof one-letter abbreviations for proline, glutamic acid, serine,threonine pI isoelectric point RIP radioimmunoprecipitation RIPAradioimmunoprecipitation assay SAC protein A-Staphylococcus aureus cellsSDRE serum dose response element SDS sodium dodecyl sulfate SDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresis SP-RIAsolid-phase radioimmunoassay SSPE NaCl (0.18 M), sodium phosphate (0.01M), EDTA (0.001 M) TBE Tris-borate/EDTA electrophoresis buffer TCAtrichloroacetic acid TC media tissue culture media TMBtetramethylbenzidine Tris tris (hydroxymethyl) aminomethane μCimicrocurie μg microgram μl microliter μM micromolar VSV vesicularstomatitis virus X-MLV xenotropic murine leukemia virus

Cell Lines

[0041] The following cell lines were used in the experiments hereindescribed: HeLa K standard type of HeLa cells; aneuploid,epithelial-like cell line isolated from a human cervical adenocarcinoma[Gey et al., Cancer Res., 12: 264 (1952); Jones et al., Obstet.Gynecol., 38: 945-949 (1971)] obtained from Professor B. Korych,[Institute of Medical Microbiology and Immunology, Charles University;Prague, Czech Republic] HeLa D98/AH.2 Mutant HeLa clone that ishypoxanthine (also HeLa S) guanine phosphoribosyl transferase- deficient(HGPRT⁻) kindly provided by Eric J. Stanbridge [Department ofMicrobiology, College of Medicine, University of California, Irvine, CA(USA)] and reported in Stanbridge et al., Science, 215: 252-259 (Jan.15, 1982); parent of hybrid cells H/F-N and H/F-T, also obtained from E.J. Stanbridge. NIH-3T3 murine fibroblast cell line reported in Aaronson,Science, 237: 178 (1987). T47D cell line derived from a human mammarycarcinoma [Keydar et al., Eur. J. Cancer, 15: 659-670 (1979)]; kindlyprovided by J. Keydar [Haddasah Medical School; Jerusalem, Israel] T24cell line from urinary bladder carcinoma [Bubenik et al., Int. J.Cancer, 11: 765-773 (1973)] kindly provided by J. Bubenik [Institute ofMolecular Genetics, Czechoslovak Academy of Sciences; Prague, CzechRepublic] HMB2 cell line from melanoma [Svec et al., Neoplasma, 35:665-681 (1988)] HEF human embryo fibroblasts [Zavada et al., Nature NewBiology, 240: 124-125 (1972)] SIRC cell line from rabbit cornea (controland X-MLV-infected) [Zavada et al., Virology, 82: 221-231 (1977)] Verocells African green monkey cell line [Zavada et al. (1977)] myeloma cellmyeloma cell line used as a fusion parent line NS-0 in production ofmonoclonal antibodies [Galfre and Milstein, Methods Enzymol., 73: 3-46(1981)] SK-Mel 1477 human melanoma cell line kindly provided by K. E.Hellstrom [Division of Tumor Immunology, Fred Hutchins Cancer ResearchCenter; Seattle, Washington (USA)] XC cells derived from a ratrhabdomyosarcoma induced with Rous sarcoma virus-induced rat sarcoma[Svoboda, J., Natl. Cancer Center Institute Monograph No. 17, IN:“International Conference on Avian Tumor Viruses” (J. W. Beard ed.), pp.277-298 (1964)], kindly provided by Jan Svoboda [Institute of MolecularGenetics, Czechoslovak Academy of Sciences; Prague, Czech Republic]; andRat 2-Tk⁻ a thymidine kinase deficient cell line, kindly provided by L.Kutinova [Institute of Sera and Vaccines; Prague, Czech Republic] CGL1H/F-N hybrid cells (HeLa D98/AH.2 derivative) CGL2 H/F-N hybrid cells(HeLa D98/AH.2 derivative) CGL3 H/F-T hybrid cells (HeLa D98/AH.2derivative) CGL4 H/F-T hybrid cells (HeLa D98/Ah.2 derivative)

Nucleotide and Amino Acid Sequence Symbols

[0042] The following symbols are used to represent nucleotides herein:Base Symbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

[0043] There are twenty main amino acids, each of which is specified bya different arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention is usedherein to identify said amino acids, as, for example, in FIGS. 1A-B andFIG. 15, respectively, as follows: Amino 3 Ltr. 1 Ltr. acid name Abbrev.Abbrev. Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid AspD Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1A-1B provides the nucleotide sequence for a MN cDNA [SEQ.ID. NO.: 1] clone isolated as described herein and the predicted aminoacid sequence [SEQ. ID. NO.: 2] encoded by the cDNA. That sequence datahas been sent to the EMBL Data Library in Heidelberg, Germany and isavailable under Accession No. X66839.

[0045]FIG. 2 provides SDS-PAGE and immunoblotting analyses ofrecombinant MN protein expressed from a pGEX-3× bacterial expressionvector. Two parallel samples of purified recombinant MN protein (twentyμg in each sample) were separated by SDS-PAGE on a 10% gel. One sample(A in FIG. 2) was stained with Coomassie brilliant blue; whereas theother sample (B) was blotted onto a Hybond C membrane [Amersham;Aylesbury, Bucks, England]. The blot was developed by autoradiographywith ¹²⁵I-labeled Mab M75.

[0046]FIG. 3 illustrates inhibition of p54/58 expression by antisenseoligodeoxynucleotides (ODNs). HeLa cells cultured in overcrowdedconditions were incubated with (A) 29-mer ODNI [SEQ. ID. NO.: 3]; (B)19-mer ODN2 [SEQ. ID. NO.: 4]; (C) both ODNI and ODN2; and (D) withoutODNS. Example 10 provides details of the procedures used.

[0047]FIG. 4 shows the results of Northern blotting of MN mRNA in humancell lines. Total RNA was prepared from the following cell lines: HeLacells growing in dense (A) and sparse (B) culture; (C)H/F-N; (D) and (E)H/F-T; and (F) human embryo fibroblasts. Example 11 details theprocedure and results.

[0048]FIG. 5 illustrates the detection of the MN gene in genomic DNAs bySouthern blotting. Chromosomal DNA digested by PstI was as follows: (A)chicken; (B) bat; (C) rat; (D) mouse; (E) feline; (F) pig; (G) sheep;(H) bovine; (I) monkey; and (J) human HeLa cells. The procedures usedare detailed in Example 12.

[0049]FIG. 6 graphically illustrates the expression of MN- andMX-specific proteins in human fibroblasts (F), in HeLa cells (H) and inH/F-N and H/F-T hybrid cells and contrasts the expression in MX-infectedand MX-uninfected cells. Example 5 details the procedures and results.

[0050]FIG. 7 (discussed in Example 5) provides immunoblots of MNproteins in fibroblasts (FIBR) and in HeLa K, HeLa S, H/F-N and H/F-Thybrid cells.

[0051]FIG. 8 (discussed in Example 6) shows immunoblots of MN proteinsin cell culture extracts prepared from the following: (A) MX-infectedHeLa cells; (B) human fibroblasts; (C) T24; (D) T47D; (E) SK-Mel 1477;and (F) HeLa cells uninfected with MX. The symbols +ME and ◯ ME indicatethat the proteins were separated by PAGE after heating in a samplebuffer, with and without 3% mercaptoethanol (ME), respectively.

[0052]FIG. 9 (discussed in Example 6) provides immunoblots of MNproteins from human tissue extracts. The extracts were prepared from thefollowing: (A) MX-infected HeLa cells; (B) full-term placenta; (C)corpus uteri; (D, M) adenocarcinoma endometrii; (E, N) carcinoma ovarii;(F, G) trophoblasts; (H) normal ovary; (I) myoma uteri; (J) mammarypapilloma; (K) normal mammary gland; (L) hyperplastic endometrium; (O)cervical carcinoma; and (P) melanoma.

[0053]FIG. 10 (discussed in Example 7) provides immunoblots of MNproteins from (A) MX-infected HeLa cells and from (B) Rat2-Tk⁻cells.(+ME and 0 ME have the same meanings as explained in the legend to FIG.8.) FIG. 11 (discussed in Example 8) graphically illustrates the resultsfrom radioimmunoprecipitation experiments with ¹²⁵I-GEX-3X-MN proteinand different antibodies. The radioactive protein (15×10³ cpm/tube) wasprecipitated with ascitic fluid or sera and SAC as follows: (A) asciteswith MAb M75; (B) rabbit anti-MaTu serum; (C) normal rabbit serum; (D)human serum L8; (E) human serum KH; and (F) human serum M7.

[0054]FIG. 12 (discussed in Example 8) shows the results fromradioimmunoassays for MN antigen. Ascitic fluid (dilution precipitating50% radioactivity) was allowed to react for 2 hours with (A) “cold”(unlabeled) protein GEX-3X-MN, or with extracts from cells as follows:(B) HeLa+MX; (C) Rat-2Tk⁻; (D) HeLa; (E) rat XC; (F) T24; and (G) HEF.Subsequently ¹²⁵I-labeled GEX-3X-MN protein (25×10³ cpm/tube) was addedand incubated for an additional 2 hours. Finally, the radioactivity toMAb M75 was adsorbed to SAC and measured.

[0055]FIG. 13 (discussed in Example 9) provides results ofimmunoelectron and scanning microscopy of MX-uninfected (control) andMX-infected HeLa cells. Panels A-D show ultrathin sections of cellsstained with MAb M75 and immunogold; Panels E and F are scanningelectron micrographs of cells wherein no immunogold was used. Panels Eand F both show a terminal phase of cell division. Panels A and E are ofcontrol HeLa cells; panels B, C, D and F are of MX-infected HeLa cells.The cells shown in Panels A, B and C were fixed and treated with M75 andimmunogold before they were embedded and sectioned. Such a procedureallows for immunogold decoration only of cell surface antigens. Thecells in Panel D were treated with M75 and immunogold only once they hadbeen embedded and sectioned, and thus antigens inside the cells couldalso be decorated.

[0056]FIG. 14 compares the results of immunizing baby rats to XC tumorcells with rat serum prepared against the fusion protein MN glutathioneS-transferase (GEX-3X-MN) (the IM group) with the results of immunizingbaby rats with control rat sera (the C group). Each point on the graphrepresents the tumor weight of a tumor from one rat. Example 14 detailsthose experiments.

[0057]FIG. 15 shows a complete nucleotide sequence of a MN cDNA [SEQ.ID. NO.: 5]. Also shown is the deduced amino acid sequence [SEQ. ID.NO.: 6]. The polyadenylation signal (AATAAA) and the mRNA instabilitymotif (ATTTA) are underlined. The amino acid residues of the putativesignal peptide as well as the membrane-spanning segment are italicized.The N-glycosylation site and the putative nuclear localization signalare denoted by squares and asterisks, respectively. The S/TPXX elementsare indicated with open circles.

[0058]FIG. 16 is a restriction map of the full-length MN cDNA. The openreading frame is shown as an open box. The thick lines below therestriction map illustrate the sizes and positions of two overlappingcDNA clones. The horizontal arrows indicate the positions of primers R1[SEQ. ID. NO.: 7) and R2 (SEQ. ID. NO.: 8] used for the 5′ end RACE.Relevant restriction sites are BamHI (B), EcoRV (V), EcoRI (E), PstI(Ps), PvuII (Pv).

[0059]FIG. 17 shows a restriction analysis of the MN gene. Genomic DNAfrom HeLa cells was cleaved with the following restriction enzymes:EcoRI (1), EcoRV (2), HindIII (3), KpnI (4), NcoI (5), PstI (6), andPvuII (7), and then analyzed by Southern hybridization under stringentconditions using MN cDNA as a probe.

[0060]FIG. 18 provides a hydrophilicity profile of the MN protein shownin FIG. 15. The profile was computed using an average group length of 6amino acids.

[0061]FIG. 19(a) shows an alignment of HCA plots derived from MN, humanCA VI (hCA) and CA II (CA2). A one-letter code is used for all aminoacids with exception of P (stars), G (diamond-shaped symbol), T and S(open and dotted squares, respectively). Strands D, E, F and G areessential for the structural core of CA. Topologically conservedhydrophobic amino acids are shaded (in hCA VI and MN). Ligands of thecatalytic zinc ion (His residues) are indicated by arrowheads.

[0062]FIG. 19(b) presents a stereoview of the CA II three-dimensionalstructure illustrating a superposition of the complete CA II structure(thin ribbon) with the structure which is well conserved in MN (openthick ribbon).

[0063]FIG. 19(c) presents an HCA comparison of thebasic/helix-loop-helix/zipper domains of Max and Myf-3 with theN-terminal part of MN. The 3D structure of Max is indicated above itsplot with delineation of the segments b: basic, h1: helix 1, L: loop,h2: helix 2, z: zipper. The I(6×)Y motif is shaded within helix 2.

[0064]FIG. 19(d) schematically represents the sequence elements andstructural domains predicted from the deduced amino acid sequence of MN.

[0065]FIG. 20 schematically represents an MN promoter region. Theconsensus sequences are as follows: CAT-CCAAT; TATA-ATAAATATA;AP2-1-GSSWSCC; AP2-YSSCCMNSSS [SEQ. ID. NO.: 19]; SP1-KMGGCCKRRY [SEQ.ID. NO.: 20]; p53-RRRCWWGYYY [SEQ. ID. NO.: 21]; and SDRE-CACCSCAC.

[0066]FIG. 21 schematically represents the 5′ MN genomic region of an MNgenomic clone.

[0067]FIG. 22(a) shows the zinc-binding activity of MN protein extractedfrom HeLa cells persistently infected with LCMV. Samples wereconcentrated by immunoprecipitation with Mab M75 before loading (A).,and after elution from ZnC₁₋₂-saturated (B) or ZnCl₂-free Fast-Flowchelating Sepharase column (c). Immunoprecipitates were analyzed byWestern blotting using iodinated M75 antibody.

[0068]FIG. 22(b) shows MN protein binding to DNA-cellulose. Proteinsextracted from LCMV-infected HeLa cells were incubated withDNA-cellulose (A). Proteins that bound to DNA-cellulose in the presenceof ZnCl₂ and absence of DTT (B), in the presence of both ZnCl₂ and DTT(C), and in the absence of both ZnCl₂ and DTT (D) were eluted, and allsamples were analyzed as above.

[0069]FIG. 22(c) shows the results of endoglycosidase H and F digestion.MN protein immunoprecipitated with Mab M75 was treated with Endo F (F)and Endo H(H). Treated (+) and control samples (−) were analyzed byWestern blotting as above.

[0070]FIG. 23 shows the morphology and growth kinetics of control (a, c,e and g) and MN-expressing (b, d, f and h) NIH 3T3 cells. Themicrographs are of methanol fixed and Giemsa stained cells at amagnification ×100. Cells were grown to confluency (a, b), or asindividual colonies in Petri dishes (c, d) and in soft agar (e, f). The(g) and (h) graphs provide growth curves of cells cultured in DMEMmedium containing respectively, 10% and 1% FCS. The mean values oftriplicate determinations were plotted against time.

[0071]FIG. 24 illustrates flow cytometric analyses of asynchromous cellpopulations of control and MN cDNA-transfected NIH 3T3 cells.

[0072]FIG. 25a-b is the complete sequence of an MN genomic clone of thisinvention [SEQ. ID. NO.: 23]. It is 5052 nucleotides long with thetranscription start site at position 3534 (starting with ACAGTCA . . .). The presumed promoter region is about 300 to 400 nucleotides upstreamof the transciption start site.

DETAILED DESCRIPTION

[0073] As demonstrated herein MaTu was found to be a two-componentsystem. One part of the complex, exogenous MX, is transmissible, and ismanifested by a protein, p58X, which is a cytoplasmic antigen whichreacts with some natural sera, of humans and of various animals. Theother component, MN, is endogenous to human cells. The level of MNexpression has been found to be considerably increased in the presenceof the MaTu-MX transmissible agent, which has been now identified aslymphocytic choriomeningitis virus (LCMV) which persistently infectsHeLa cells.

[0074] MN is a cellular gene, showing only very little homology withknown DNA sequences. It is rather conservative and is present as asingle copy gene in the chromosomal DNA of various vertebrates.Described herein is the cloning and sequencing of the MN cDNA, and thegenetic engineering of MN proteins—such as the GEX-3X-MN and MN 20-19proteins. The recombinant MN proteins can be conveniently purified byaffinity chromatography.

[0075] MN is manifested in HeLa cells by a twin protein, p54/58N, thatis localized on the cell surface and in the nucleus. Immunoblots using amonoclonal antibody reactive with p54/58N (MAb M75) revealed two bandsat 54 kd and 58 kd. Those two bands may correspond to one type ofprotein that differs by glycosylation pattern or by how it is processed.(Both p54N and p58N are glycosylated with oligosaccharidic residuescontaining mannose, but only p58N also contains glucosamine.) Herein,the phrase “twin protein” indicates p54/58N.

[0076] MN is absent in rapidly growing, sparse cultures of HeLa, but isinducible either by keeping the cells in dense cultures or, moreefficiently, by infecting them with MX (LCMV). Those inducing factorsare synergistic. p54/58N and not p58X is associated with virions ofvesicular stomatitis virus (VSV), reproduced in MaTu-infected HeLa.Whereas the twin protein p54/58N is glycosylated and forms oligomerslinked by disulfidic bonds, p58X is not glycosylated and does not formS-S-linked oligomers.

[0077] VSV assembles p54/58N into virions in HeLa cells, indicating thatthe twin protein is responsible for complementation of VSV G-proteinmutants and for formation of VSV(MaTu) pseudotypes. As only envelopedviruses provide surface glycoproteins for the formation of infectious,functioning pseudotypes, which can perform such specific functions asadsorption and penetration of virions into cells [Zavada, J., J. Gen.Virol., 63: 15-24 (1982)], that observation implies that the MN genebehaves as a quasi-viral sequence.

[0078] The surface proteins of enveloped viruses, which participate inthe formation of VSV pseudotypes, are glycosylated as is the MN twinprotein, p54/58N. MN proteins also resemble viral glycoproteins in theformation of oligomers (preferably tri- or tetramers); sucholigomerization, although not necessarily involving S-S bonds(disulfidic bonds), is essential for the assembly of virions [Kreis andLodish, Cell. 46: 929-937 (1986)]. The disulfidic bonds can be disruptedby reduction with 2-mercaptoethanol.

[0079] As reported in Pastorekova et al., Virology. 187: 620-626 (1992),after reduction with mercaptoethanol, p54/58N from cell extracts or fromVSV looks very similar on immunoblot. Without reduction, in cellextracts, it gives several bands around 150 kd, suggesting that thecells may contain several different oligomers (probably with a differentp54:p58 ratio), but VSV selectively assembles only one of them, with amolecular weight of about 153 kd. That oligomer might be a trimer, orrather a tetramer, consisting of 54 kd and 58 kd proteins. The equimolarratio of p54:p58 in VSV virions is indicated by approximately the samestrength of 54 kd and 58 kd bands in a VSV sample analyzed underreducing conditions.

[0080] The expression of MN proteins appears to be diagnostic/prognosticfor neoplastic disease. The MN twin protein, p54/58N, was found to beexpressed in HeLa cells and in Stanbridge's tumorigenic (H/F-T) hybridcells [Stanbridge et al., Somatic Cell Genet, 7: 699-712 (1981); andStanbridge et al., Science. 215: 252-259 (1982)] but not in fibroblastsor in non-tumorigenic (H/F-N) hybrid cells [Stanbridge et al., id.]. Inearly studies, MN proteins were found in immunoblots prepared from humanovarian, endometrial and uterine cervical carcinomas, and in some benignneoplasias (as mammary papilloma) but not from normal ovarian,endometrial, uterine or placental tissues. Example 13 details furtherresearch on MN gene expression wherein MN antigen, as detected byimmunohistochemical staining, was found to be prevalent in tumor cellsof a number of cancers, including cervical, bladder, head and neck, andrenal cell carcinomas among others. Further, the immunohistochemicalstaining experiments of Example 13 show that among normal tissuestested, only normal stomach tissues showed routinely and extensively thepresence of MN antigen. MN antigen is further shown herein to be presentsometimes in morphologically normal-appearing areas of tissue specimensexhibiting dysplasia and/or malignancy.

[0081] In HeLa cells infected with MX, observed were conspicuousultrastructural alterations, that is, the formation of abundantfilaments on cell surfaces and the amplification of mitochondria. Usingan immunogold technique, p54/58N was visualized on the surface filamentsand in the nucleus, particularly in the nucleoli. Thus MN proteinsappear to be strongly correlated with tumorigenicity, and do not appearto be produced in general by normal non-tumor cells.

[0082] Examples herein show that MX and MN are two different entities,that can exist independently of each other. MX (LCMV) as an exogenous,transmissible agent can multiply in fibroblasts and in H/F-N hybridcells which are not expressing MN-related proteins (FIG. 6). In suchcells, MX does not induce the production of MN protein. MN protein canbe produced in HeLa and other tumor cells even in the absence of MX asshown in FIGS. 6-9. However, MX is a potent inducer of MN-relatedprotein in HeLa cells; it increases its production thirty times over theconcentration observed in uninfected cells (FIGS. 7 and 12, Table 1 inExample 8, below).

MN Gene—Cloning and Sequencing

[0083]FIGS. 1A-1B and 15 provide the nucleotide sequences for MN cDNAclones isolated as described below, respectively SEQ. ID. NOS.: 1 and 5.FIG. 25a-b provides the sequence of a MN genomic clone containing apromoter region [SEQ. ID. NO.: 23].

[0084] It is understood that because of the degeneracy of the geneticcode, that is, that more than one codon will code for one amino acid[for example, the codons TTA, TTG, CTT, CTC, CTA and CTG each code forthe amino acid leucine (leu)], that variations of the nucleotidesequences in, for example, SEQ. ID. NOS.: 1, 5, and 23 wherein one codonis substituted for another, would produce a substantially equivalentprotein or polypeptide according to this invention. All such variationsin the nucleotide sequences of the MN cDNA and complementary nucleicacid sequences are included within the scope of this invention.

[0085] It is further understood that the nucleotide sequences hereindescribed and shown in FIGS. 1A-1B, 15 and 25, represent only theprecise structures of the cDNA and genomic nucleotide sequences isolatedand described herein. It is expected that slightly modified nucleotidesequences will be found or can be modified by techniques known in theart to code for substantially similar or homologous MN proteins andpolypeptides, for example, those having similar epitopes, and suchnucleotide sequences and proteins/polypeptides are considered to beequivalents for the purpose of this invention. DNA or RNA havingequivalent codons is considered within the scope of the invention, asare synthetic nucleic acid sequences that encode proteins/polypeptideshomologous or substantially homologous to MN proteins/polypeptides, aswell as those nucleic acid sequences that would hybridize to saidexemplary sequences [SEQ. ID. NOS. 1, 5 and 23] under stringentconditions or that but for the degeneracy of the genetic code wouldhybridize to said cDNA nucleotide sequences under stringenthybridization conditions. Modifications and variations of nucleic acidsequences as indicated herein are considered to result in sequences thatare substantially the same as the exemplary MN sequences and fragmentsthereof.

[0086] Partial cDNA Clone

[0087] To find the MN gene, a lambda gt11 cDNA library from MX-infectedHeLa cells was prepared. Total RNA from MX-infected HeLa cells wasisolated by a guanidinium-thiocyanate-CsCl method [Chirgwin et al.,Biochemistry, 18: 5249 (1979)], and the mRNA was affinity separated onoligo dT-cellulose [Ausubel et al., Short Protocols in MolecularBiology, (Greene Publishing Assocs. and Wiley-Interscience; NY, USA,1989]. The synthesis of the cDNA and its cloning into lambda gt11 wascarried out using kits from Amersham, except that the EcORI-NotI adaptorwas from Stratagene [La Jolla, Calif. (USA)]. The library was subjectedto immunoscreening with Mab M75 in combination with goat anti-mouseantibodies conjugated with alkaline phosphatase. That immunoscreeningmethod is described in Young and Davis, PNAS (USA). 80: 1194-1198(1983). About 4×10⁵ primary plaques on E. coli Y1090 cells, representingabout one-half of the whole library, were screened using Hybond N+membrane [Amersham] saturated with 10 mM IPTG and blocked with 5% FCS.Fusion proteins were detected with Mab M75 in combination with goatanti-mouse antibodies conjugated with alkaline phosphatase. One positiveclone was picked.

[0088] pBluescript-MN. The positive clone was subcloned into the NotIsite of pBluescript KS [Stratagene] thereby creating pBluescript-MN. Twooppositely oriented nested deletions were made using Erase-a-Base™ kit[Promega; Madison, Wis. (USA)] and sequenced by dideoxy method with a T7sequencing kit [Pharmacia; Piscataway, N.J. (USA)]. The sequencingshowed a partial cDNA clone, the insert being 1397 bp long. Thatsequence is shown in FIG. 1A-1B. The sequence comprises a large 1290 bpopen reading frame and 107 bp 3′ untranslated region containing apolyadenylation signal (AATAAA). Another interesting feature of thesequence is the presence of a region contributing to instability of themRNA (AUUUA at position 1389) which is characteristic for mRNAs of someoncogenes and lymphokines [Shaw and Kamen, Cell. 46: 659-667 (1986)].However, the sequence surrounding the first ATG codon in the openreading frame (ORF) did not fit the definition of a translational startsite. In addition, as follows from a comparison of the size of the MNclone with that of the corresponding mRNA in a Northern blot (FIG. 4),the cDNA was missing about 100 bp from the 5′ end of its sequence.

[0089] Full-Length cDNA Clone

[0090] Attempts to isolate a full-length clone from the original cDNAlibrary failed. Therefore, we performed a rapid amplification of cDNAends (RACE) using MN-specific primers, R1 and R2, derived from the 5′region of the original cDNA clone. The RACE product was inserted intopBluescript, and the entire population of recombinant plasmids wassequenced with an MN-specific primer ODN1. In that way, we obtained areliable sequence at the very 5′ end of the MN cDNA as shown in FIG. 15[SEQ. ID. NO.: 5].

[0091] Specifically, RACE was performed using 5′ RACE System [GIBCO BRL;Gaithersburg, Md. (USA)] as follows. 1 μg of mRNA (the same-as above)was used as a template for the first strand cDNA synthesis which wasprimed by the MN-specific antisense oligonucleotide, R1(5′-TGGGGTTCTTGAGGATCTCCAGGAG-3′) [SEQ. ID. NO.: 7]. The first strandproduct was precipitated twice in the presence of ammonium acetate and ahomopolymeric C tail was attached to its 3′ end by TdT. Tailed cDNA wasthen amplified by PCR using a nested primer, R2(5′-CTCTAACTTCAGGGAGCCCTCTTCTT-3′) [SEQ. ID. NO.: 8] and an anchorprimer that anneals to the homopolymeric tail(5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGI IGGGIIGGGIIG-3′) [SEQ. ID. NO.:9]. Amplified product was digested with BamHI and SalI restrictionenzymes and cloned into pBluescript II KS plasmid. After transformation,plasmid DNA was purified from the whole population of transformed cellsand used as a template for the sequencing with the MN-specific primerODN1 [SEQ. ID. NO.: 3; a 29-mer, the sequence for which is shown inExample 10].

[0092] The full-length MN cDNA sequence is 1519 base pairs (bp) long(FIG. 15). It contains a single ORF of 1400 bp, starting at position 12,with an ATG codon that is in a good context (GCGCATGG) with the ruleproposed for translation initiation [Kozak, J. Cell. Biol. 108: 229-241(1989)]. The AT rich 3′ untranslated region contains, as indicatedabove, a polyadenylation signal (AATAAA) preceding the end of the cDNAby 10 bp. Surprisingly, the sequence from the original clone as well asfrom four additional clones obtained from the same cDNA library did notreveal any poly(A) tail. Moreover, also as indicated above, justdownstream of the poly(A) signal we found an ATTTA motif that is thoughtto contribute to mRNA instability (Shaw and Kamen, supra). This factraised possibility that the poly (A) tail is missing due to the specificdegradation of the MN mRNA.

[0093] Genomic Clone

[0094] To study MN regulation, an MN genomic clone was isolated from ahuman cosmid library prepared from fetal brain using both the MN cDNAprobe and the MN-specific primers derived from the 5′ end of the cDNA[SEQ. ID. NOS.: 3 and 4; ODNI AND ODN2; see Example 10]. The sequencefor that genomic clone is shown in FIG. 25 [SEQ. ID. NO.: 23]. Sequenceanalysis revealed that the genomic clone covers a region upstream fromthe MN transcription start site and ending with the BamHI restrictionsite localized inside the MN cDNA. Other MN genomic clones can besimilarly isolated.

[0095] The promoter region is GC-rich and contains one putative TATA-box578 bp upstream from the transcription start. The promoter containsseveral consensus sequences for binding sites of regulatory elements,including two p53 sites, two AP-2 sites, an AP-1 site, a SP-1 site, anda SDRE site. FIG. 20 provides a schematic of the MN promoter region, andFIG. 21 provides a schematic of the 5′ MN genomic region.

[0096] Interestingly, the 5′ end region of the isolated genomic clone isstrongly homologous to the 5′ long terminal repeat (LTR) of humanendogenous retroviruses HERV K. As shown in FIG. 21, there is no codingsequence, only two Alu repeats, between the LTR-like region and thepromoter. That fact suggests that the LTR, although not necessarilybelonging to MN, may provide an enhancer for MN transcription.

[0097] Deduced Amino Acid Sequences

[0098] The open reading frame of the MN cDNA clone shown in FIG. 1A-1Bencodes a putative protein of 429 amino acids with a calculatedmolecular weight of about 48 kd. The hydrophilicity profile reveals ahydrophobic sequence of amino acids (at positions 371-395) probablyrepresenting the region spanning the plasma membrane and containing alsoa potential cleavage signal. The profile fits well with the observationthat p54/58N proteins are localized on the cell membrane. There are noPEST regions in the MN amino acid sequence, suggesting that the productof the MN gene is a stable long-lived protein [Rogers et al., Science,234: 364-368 (1986)]. Such a feature explains our experience withinefficient metabolic labeling of p54/58N. The deduced amino acidsequence displays also other features namely, 10 potentialphosphorylation and 7 myristylation sites, and 3 antigenic determinants.

[0099] The deduced amino acid sequence from the partial cDNA sequenceshown in FIG. 1A-1B can be compared to that shown in FIG. 15 from thefull-length cDNA. The partial sequence is missing the N-terminal 37amino acids—the putative signal peptide. The ORF of the MN cDNA shown inFIG. 15 has the coding capacity for a 466 amino acid protein with acalculated molecular weight of 51.5 kd. As assessed by amino acidsequence analysis, the deduced primary structure of the MN protein canbe divided into four distinct regions. The initial hydrophobic region of37 amino acids (AA) corresponds to a signal peptide. The mature proteinhas an N-terminal part of 377 AA, a hydrophobic transmembrane segment of20 AA and a C-terminal region of 32 AA. The overall amino acidcomposition is rather basic, with a predicted isoelectric point of 8.92.The MN protein is rich in leucine (11.16%), proline (10.3%), alanine(9.44%), arginine (9.23%), and serine (9.01%).

[0100] More detailed insight into MN protein primary structure disclosedthe presence of several consensus sequences. One potentialN-glycosylation site was found at position 345 of FIG. 15, and aputative nuclear localization signal composed of a stretch of basicamino acids RRARKK [Blank et al., EMBO J., 10: 4159-4167 (1991); Wangand Reed, Nature, 364: 121-126 (1993)] was recognized in the middle ofthe protein, at position 279-284 of FIG. 15. These features, togetherwith the predicted membrane-spanning region mentioned above, areconsistent with the results, in which MN was shown to be anN-glycosylated protein localized both in the plasma membrane and in thenucleus. MN protein sequence deduced from cDNA was also found to containsix S/TPXX sequence elements [SEQ. ID. NOS.: 25 AND 26] (one of them isin the signal peptide) defined by Suzuki, J. Mol. Biol. 207: 61-84(1989) as motifs frequently found in gene regulatory proteins. However,only two of them are composed of the suggested consensus amino acids.

Sequence Similarities and HCA

[0101] Computer analysis of the MN cDNA sequence was carried out usingDNASIS and PROSID (Pharmacia Software packages). GenBank, EMBL, ProteinIdentification Resource and SWISS-PROT databases were searched for allpossible sequence similarities. In addition, a search for proteinssharing sequence similarities with MN was performed in the MIPS databankwith the FastA program [Pearson and Lipman, PNAS (USA), 85: 2444(1988)].

[0102] The MN gene was found to clearly be a novel sequence derived fromthe human genome. Searches for amino acid sequence similarities inprotein databases revealed as the closest homology a level of sequenceidentity (44% of 170 AAs) between the central part of the MN protein[AAs 221-390 of FIG. 15 (SEQ. ID. NO.: 6)] and carbonic anhydrases (CA).However, the overall sequence homology between the cDNA MN sequence andcDNA sequences encoding different CA isoenzymes is in a homology rangeof 48-50% which is considered by ones in the art to be low. Therefore,the MN cDNA sequence is not closely related to any CA cDNA sequences.

[0103] Only very closely related sequences having a homology of at least80-90% would hybridize to each other under stringent conditions. Asequence comparison of the MN cDNA sequence shown in FIG. 1A-1B and acorresponding cDNA of the human carbonic anhydrase II (CA II) showedthat there are no stretches of identity between the two sequences thatwould be long enough to allow for a segment of the CA II cDNA sequencehaving 50 or more nucleotides to hybridize under stringent hybridizationconditions to the MN cDNA or vice versa.

[0104] Although MN deduced amino acid sequences show some homology toknown carbonic anhydrases, they differ from them in several repects.Seven carbonic anhydrases are known [Dodgson et al. (eds.), The CarbonicAnhydrases, (Plenum Press; New York/London (1991)]. All of the knowncarbonic anhydrases are proteins of about 30 kd, smaller than thep54/58N-related products of the MN gene. Further, the carbonicanhydrases do not form oligomers as do the MN-related proteins.

[0105] HCA. Hydrophobic Cluster Analysis (HCA) was used as a verysensitive method to detect similarities in the secondary and tertiaryfolding of protein domains even if the sequence homology is low.[Lemesle-Varloot et al., Biochimie, 72: 555 (1990); Thoreau et al., FEBSLett. 282: 26 (1991); Gaboriaud et al., FEBS Lett. 224: 189 (1987).]Comparison of the HCA plots for MN, human CA VI and CA II [FIG. 19(a)]showed that only the middle and C-terminal part of CA are highlyconserved in MN with the conserved zinc binding site and the enzyme'sactive center. A 44% sequence identity and 87% HCA score was calculatedbetween MN and CA VI isoenzyme. [Aldred et al., Biochemistry. 30: 569(1991).] Those values are within the range of those observed for closelyrelated structures [Lemesle-Varloot et al., supra].

[0106] HCA was also used in combination with the one-dimensionalanalysis programs, to analyze the N-terminal part of MN. When screenedagainst the MIPS sequence databank using the FastA program [Pearson andLipman, supra], the N-terminal segment of MN (AAs 38-114 of SEQ. ID.NO.: 6) repeatedly matched helical protein domains involved in theregulation of gene expression (i.e., transforming protein Myb, accessionnumber SO4897; myogenic determination factor Myf-3, accession numberSO6947; transcriptional activator YAB, accession number JE0416;translational activator PET127, accession number S17029, etc.). HCAsuggested [FIG. 19(c)] that although those hits were of low levelsequence identity, they might be structurally valid.

[0107] The most significant is a predicted structural similarity of MNto members of the HLH protein family—represented by Myf-3 and Maxprotein. The conserved I(6×)Y motif is revealed in FIG. 19(c), a motifthat is a common characteristic of an HLH protein dimerization domain[Ferre-D'Amare et al., Nature. 363: 38 (1993)]. The region between theCA-like domain and the putative HLH (covering AAs 120-220 of SEQ. ID.NO.: 6) domain is rich in imperfect repeats of Ser (15%), Pro (16%), Glyand acidic residues with few hydrophobic amino acids, resembling, thus,an activation region of transcription factors. [Lautenberger et al.,Oncogene. 7: 1713 (1992).]

[0108] In experiments, the results for which are shown in FIG. 22(a), itwas determined that MN protein is able to bind zinc cations, as shown byaffinity chromatography using Zn-charged chelating sepharose. MN proteinimmunoprecipitated from HeLa cells by Mab M75 was found to have weakcatalytic activity of CA. The CA-like domain of MN has a structuralpredisposition to serve as a binding site for small soluble domains.Thus, MN protein could mediate some kind of signal transduction.

[0109] MN protein from LCMV-infected HeLA cells was shown by using DNAcellulose affinity chromatography [FIG. 22(b)] to bind to immobilizeddouble-stranded salmon sperm DNA. The binding activity required both thepresence of zinc cations and the absence of a reducing agent in thebinding buffer.

MN Twin Protein

[0110] The possibility that the 4 kd difference between the molecularweights of the two MN proteins is caused by different glycosylation wasruled out, since after in vitro treatment with endoglycosidases H and F,respectively, both peptide portions lost about 3 kd in weight. Thisresult indicates, in addition, that the molecular weight of the smaller54 kd MN protein without its 3 kd sugar moiety, roughly corresponds tothe molecular weight of MN calculated from the full-length cDNA. Westernblot analysis of MN proteins from cervical carcinoma and normal stomachshows that in both tissues MN protein consists of two 54 and 58 kdpeptide portions.

[0111] To determine whether both p54/58N proteins were encoded by onegene, antisense ODNs were used to inhibit specifically MN geneexpression. [Such use of antisense ODNs is reviewed in Stein and Cohen,Cancer Res. 48: 2659-2668 (1988).] Those experiments are detailed inExample 10. The findings indicated that cultivation of HeLa cells withODNs resulted in a considerable inhibition of p54/58N synthesis, whereasthe amount of different HeLa cell proteins produced remainedapproximately the same. Further, and importantly, on immunoblotting, thespecific inhibition by ODNs affected both of the p54/58N proteins (FIG.3). Thus, it was concluded that the MN gene that was cloned codes forboth of the p54/58N proteins in HeLa cells.

MN Proteins and/or Polypeptides

[0112] The phrase “MN proteins and/or polypeptides” (MNproteins/polypeptides) is herein defined to mean proteins and/orpolypeptides encoded by an MN gene or fragments thereof. Exemplary andpreferred MN proteins according to this invention have the deduced aminoacid sequences shown in FIGS. 1A-1B and 15. Preferred MNproteins/polypeptides are those proteins and/or polypeptides that havesubstantial homology with the MN proteins shown in FIGS. 1A-1B and 15.For example, such substantially homologous MN proteins/polypeptides arethose that are reactive with the MN-specific antibodies of thisinvention, preferably the Mabs M75, MN12 and MN7 or their equivalents.

[0113] A “polypeptide” is a chain of amino acids covalently bound bypeptide linkages and is herein considered to be composed of 50 or lessamino acids. A “protein” is herein defined to be a polypeptide composedof more than 50 amino acids.

[0114] MN proteins exhibit several interesting features: cell membrane,and at the same time, nuclear localization (similar to E6 protein ofHPV16), cell density dependent expression in HeLa cells, correlationwith the tumorigenic phenotype of HeLa x fibroblast somatic cellhybrids, and expression in several human carcinomas among other tissues.As demonstrated herein, for example, in Example 13, MN protein can befound directly in tumor tissue sections but not in general incounterpart normal tissues (exceptions noted infra in Example 13 as innormal stomach tissues). MN is also expressed sometimes inmorphologically normal appearing areas of tissue specimens exhibitingdysplasia and/or malignancy. Taken together, these features suggest apossible involvement of MN in the regulation of cell proliferation,differentiation and/or transformation.

[0115] It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase “MN antigen” is used herein to encompass MN proteinsand/or polypeptides.

[0116] It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention.

[0117] The MN proteins and polypeptides of this invention can beprepared in a variety of ways according to this invention, for example,recombinantly, synthetically or otherwise biologically, that is, bycleaving longer proteins and polypeptides enzymatically and/orchemically. A preferred method to prepare MN proteins is by arecombinant means. Particularly preferred methods of recombinantlyproducing MN proteins are described below for the GEX-3X-MN and MN 20-19proteins.

Recombinant Production of MN Proteins and Polypeptides

[0118] A representative method to prepare the MN proteins shown in FIGS.1A-1B and 15 or fragments thereof would be to insert the appropriatefragment of MN cDNA into an appropriate expression vector as exemplifiedbelow. The fusion protein GEX-3X-MN expressed from XL1-Blue cells isnonglycosylated. Representative of a glycosylated, recombinantlyproduced MN protein is the MN 20-19 protein expressed from insect cells.The MN 20-19 protein was also expressed in a nonglycosylated form in E.coli using the expression plasmid pEt-22b [Novagen).

[0119] Baculovirus Expression Systems. Recombinant baculovirus expressvectors have been developed for infection into several types of insectcells. For example, recombinant baculoviruses have been developed foramong others: Aedes aegypti, Autographa californica, Bombvx mor,Drosphila melanogaster, Heliothis zea, Spodoptera frugiperda, andTrichoplusia ni [PCT Pub. No. WO 89/046699; Wright, Nature, 321: 718(1986); Fraser et al., In Vitro Cell Dev. Biol. 25: 225 (1989). Methodsof introducing exogenous DNA into insect hosts are well-known in theart. DNA transfection and viral infection procedures usually vary withthe insect genus to be transformed. See, for example, Autographa[Carstens et al., Virology. 101: 311 (1980)]; Spodoptera [Kang,“Baculovirus Vectors for Expression of Foreign Genes,” in: Advances inVirus Research. 35 (1988)]; and Heliothis (virescens) [PCT Pub. No. WO88/02030].

[0120] A wide variety of other host-cloning vector combinations may beusefully employed in cloning the MN DNA isolated as described herein.For example, useful cloning vehicles may include chromosomal,nonchromosomal and synthetic DNA sequences such as various knownbacterial plasmids such as pBR322, other E. coli plasmids and theirderivatives and wider host range plasmids such as RP4, phage DNA, suchas, the numerous derivatives of phage lambda, e.g., NB989 and vectorsderived from combinations of plasmids and phage DNAs such as plasmidswhich have been modified to employ phage DNA expression controlsequences.

[0121] Useful hosts may be eukaryotic or prokaryotic and includebacterial hosts such as E. coli and other bacterial strains, yeasts andother fungi, animal or plant hosts such as animal or plant cells inculture, insect cells and other hosts. Of course, not all hosts may beequally efficient. The particular selection of host-cloning vehiclecombination may be made by those of skill in the art after dueconsideration of the principles set forth herein without departing fromthe scope of this invention.

[0122] The particular site chosen for insertion of the selected DNAfragment into the cloning vehicle to form a recombinant DNA molecule isdetermined by a variety of factors. These include size and structure ofthe protein or polypeptide to be expressed, susceptibility of thedesired protein or polypeptide to endoenzymatic degradation by the hostcell components and contamination by its proteins, expressioncharacteristics such as the location of start and stop codons, and otherfactors recognized by those of skill in the art.

[0123] The recombinant nucleic acid molecule containing the MN gene,fragment thereof, or cDNA therefrom, may be employed to transform a hostso as to permit that host (transformant) to express the structural geneor fragment thereof and to produce the protein or polypeptide for whichthe hybrid DNA encodes. The recombinant nucleic acid molecule may alsobe employed to transform a host so as to permit that host on replicationto produce additional recombinant nucleic acid molecules as a source ofMN nucleic acid and fragments thereof. The selection of an appropriatehost for either of those uses is controlled by a number of factorsrecognized in the art. These include, for example, compatibility withthe chosen vector, toxicity of the co-products, ease of recovery of thedesired protein or polypeptide, expression characteristics, biosafetyand costs.

[0124] Where the host cell is a procaryote such as E. coli, competentcells which are capable of DNA uptake are prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by well known procedures. Transformation can also be performedafter forming a protoplast of the host cell.

[0125] Where the host used is an eucaryote, transfection methods such asthe use of a calcium phosphate-precipitate, electroporation,conventional mechanical procedures such as microinjection, insertion ofa plasmid encapsulated in red blood cell ghosts or in liposomes,treatment of cells with agents such as lysophosphatidyl-choline or useof virus vectors, or the like may be used.

[0126] The level of production of a protein or polypeptide is governedby three major factors: (1) the number of copies of the gene or DNAsequence encoding for it within the cell; (2) the efficiency with whichthose gene and sequence copies are transcribed and translated; and (3)the stability of the mRNA. Efficiencies of transcription and translation(which together comprise expression) are in turn dependent uponnucleotide sequences, normally situated ahead of the desired codingsequence. Those nucleotide sequences or expression control sequencesdefine, inter alia, the location at which an RNA polymerase interacts toinitiate transcription (the promoter sequence) and at which ribosomesbind and interact with the mRNA (the product of transcription) toinitiate translation. Not all such expression control sequences functionwith equal efficiency. It is thus of advantage to separate the specificcoding sequences for the desired protein from their adjacent nucleotidesequences and fuse them instead to known expression control sequences soas to favor higher levels of expression. This having been achieved, thenewly engineered DNA fragment may be inserted into a multicopy plasmidor a bacteriophage derivative in order to increase the number of gene orsequence copies within the cell and thereby further improve the yield ofexpressed protein.

[0127] Several expression control sequences may be employed. Theseinclude the operator, promoter and ribosome binding and interactionsequences (including sequences such as the Shine-Dalgarno sequences) ofthe lactose operon of E. coli (“the lac system”), the correspondingsequences of the tryptophan synthetase system of E. coli (“the trpsystem”), a fusion of the trp and lac promoter (“the tac system”), themajor operator and promoter regions of phage lambda (O_(L)P_(L) andO_(R)P_(R′)), and the control region of the phage fd coat protein. DNAfragments containing these sequences are excised by cleavage withrestriction enzymes from the DNA isolated from transducing phages thatcarry the lac or trp operons, dr from the DNA of phage lambda or fd.Those fragments are then manipulated in order to obtain a limitedpopulation of molecules such that the essential controlling sequencescan be joined very close to, or in juxtaposition with, the initiationcodon of the coding sequence.

[0128] The fusion product is then inserted into a cloning vehicle fortransformation or transfection of the appropriate hosts and the level ofantigen production is measured. Cells giving the most efficientexpression may be thus selected. Alternatively, cloning vechiclescarrying the lac, trp or lambda P_(L) control system attached to aninitiation codon may be employed and fused to a fragment containing asequence coding for a MN protein or polypeptide such that the gene orsequence is correctly translated from the initiation codon of thecloning vehicle.

[0129] The phrase “recombinant nucleic acid molecule” is herein definedto mean a hybrid nucleotide sequence comprising at least two nucleotidesequences, the first sequence not normally being found together innature with the second.

[0130] The phrase “expression control sequence” is herein defined tomean a sequence of nucleotides that controls and regulates expression ofstructural genes when operatively linked to those genes.

[0131] The following are representative examples of geneticallyengineering MN proteins of this invention. The descriptions areexemplary and not meant to limit the inventin in any way.

[0132] Production of Fusion Protein GEX-3X-MN

[0133] To confirm whether the partial cDNA clone codes for thep54/58N-specific protein, it was subcloned into the bacterial expressionvector pGEX-3X [Pharmacia; Upsala, Sweden], constructed to express afusion protein containing the C-terminus of glutathione S-transferase.The partial cDNA insert from the above-described pBluescript-MN wasreleased by digesting the plasmid DNA by NotI. It was then treated withS1 nuclease to obtain blunt ends and then cloned into a dephosphorylatedSmaI site of pGEX-3X [Pharmacia]. After transformation of XL1-Blue cells(E. coli strain; Stratagene] and induction with IPTG, a fusion proteinwas obtained.

[0134] The fusion protein—MN glutathione S-transferase (GEX-3X-MN) waspurified by affinity chromatography on Glutathione-S-Sepharose 4B(Pharmacia]. Twenty micrograms of the purified recombinant protein ineach of two parallel samples were separated by SDS-PAGE on a 10% gel.One of the samples (A) was stained with Coomassie brilliant blue,whereas the other (B) was blotted onto a Hybond C membrane (Amersham).The blot was developed by autoradiography with ¹²⁵I-labeled MAb M75. Theresults are shown in FIG. 2.

[0135] SDS-PAGE analysis provided an interesting result: a number ofprotein bands with different molecular weights (FIG. 2A). A similarSDS-PAGE pattern was obtained with another representative fusion proteinproduced according to this invention, beta-galactosidase-MN that wasexpressed from lambda gt11 lysogens. It appears that those patterns aredue to translation errors caused by the presence of 9 AGGAGG codontandems in the MN sequence. The use of those codons is strongly avoidedin bacterial genes because of the shortage of corresponding tRNAs. Thus,during the translation of AGGAGG tandems from foreign mRNA, +1 ribosomalframeshifts arise with a high frequency (about 50%) [Spanjaard et al.,Nuc. Acid Res., 18: 5031-5036 (1990)].

[0136] By immunoblotting, a similar pattern was obtained: all the bandsseen on stained SDS-PAGE gel reacted with the MN-specific MAb M75 (FIG.2B), indicating that all the protein bands are MN-specific. Also, thatresult indicates that the binding site for MAb M75 is on the N-terminalpart of the MN protein, which is not affected by frameshifts.

[0137] As shown in Example 8 below, the fusion protein GEX-3X-MN wasused in radioimmunoassays for MN-specific antibodies and for MN antigen.

[0138] Expression of MN 20-19 Protein

[0139] Another representative, recombinantly produced MN protein of thisinvention is the MN 20-19 protein which, when produced inbaculovirus-infected Sf9 cells [Spodoptera frugiperda cells; Clontech;Palo Alto, Calif. (USA)], is glycosylated. The MN 20-19 protein missesthe putative signal peptide (AAs 1-37) of SEQ. ID. NO.: 6 (FIG. 15), hasa methionine (Met) at the N-terminus for expression, and aLeu-Glu-His-His-His-His-His-His [SEQ. ID NO.: 22] added to theC-terminus for purification. In order to insert the portion of the MNcoding sequence for the GEX-3X-MN fusion protein into alternateexpression systems, a set of primers for PCR was designed. The primerswere constructed to provide restriction sites at each end of the codingsequence, as well as in-frame start and stop codons. The sequences ofthe primers, indicating restriction enzyme cleavage sites and expressionlandmarks, are shown below.

[0140] The SEQ. ID. NOS.: 17 and 18 primers were used to amplify the MNcoding sequence present in the pGEX-3X-MN vector using standard PCRtechniques. The resulting PCR product (termed MN 20-19) waselectrophoresed on a 0.5% agarose/1X TBE gel; the 1.3 kb band wasexcised; and the DNA recovered using the Gene Clean II kit according tothe manufacturer's instructions [Bio101; LaJolla, Calif. (USA)].

[0141] MN 20-19 and plasmid pET-22b [Novagen, Inc.; Madison, Wis. (USA)]were cleaved with the restriction enzymes NdeI and XhoI,phenol-chloroform extracted, and the appropriate bands recovered byagarose gel electrophoresis as above. The isolated fragments wereethanol co-precipitated at a vector:insert ratio of 1:4. Afterresuspension, the fragments were ligated using T4 DNA ligase. Theresulting product was used to transform competent Novablue E. coli cells[Novagen, Inc.]. Plasmid mini-preps [Magic Minipreps; Promega] from theresultant ampicillin resistant colonies were screened for the presenceof the correct insert by restriction mapping. Insertion of the genefragment into the pET-22b plasmid using the NdeI and XhoI sites added a6-histidine tail to the protein that could be used for affinityisolation.

[0142] To prepare MN 20-19 for insertion into the baculovirus expressionsystem, the MN 20-19 gene fragment was excised from pET-22b using therestriction endonucleases XbaI and PvuI. The baculovirus shuttle vectorpBacPAK8 [Clontech] was cleaved with XbaI and PacI. The desiredfragments (1.3 kb for MN 20-19 and 5.5 kb for pBacPAK8) were isolated byagarose gel electrophoresis, recovered using Gene Clean II, andco-precipitated at an insert:vector ratio of 2.4:1.

[0143] After ligation with T4 DNA ligase, the DNA was used to transformcompetent NM522 E. coli cells (Stratagene). Plasmid mini-preps fromresultant ampicillin resistant colonies were screened for the presenceof the correct insert by restriction mapping. Plasmid DNA from anappropriate colony and linearized BacPAK6 baculovirus DNA [Clontech]were used to transform Sf9 cells by standard techniques. Recombinationproduced BacPAK viruses carrying the MN 20-19 sequence. Those viruseswere plated onto Sf9 cells and overlaid with agar.

[0144] Plaques were picked and plated onto Sf9 cells. The conditionedmedia and cells were collected. A small aliquot of the conditioned mediawas set aside for testing. The cells were extracted with PBS with 1%Triton X100.

[0145] The conditioned media and the cell extracts were dot blotted ontonitrocellulose paper. The blot was blocked with 5% non-fat dried milk inPBS. Mab M75 were used to detect the MN 20-19 protein in the dot blots.A rabbit anti-mouse Ig-HRP was used to detect bound Mab M75. The blotswere developed with TMB/H₂O₂ with a membrane enhancer [KPL;Gaithersburg, Md. (USA)]. Two clones producing the strongest reaction onthe dot blots were selected for expansion. One was used to produce MN20-19 protein in High Five cells [Invitrogen Corp., San Diego, Calif.(USA); BTI-TN-5BI-4; derived from Trichoplusia ni egg cell homogenate].MN 20-19 protein was purified from the conditioned media from the virusinfected High Five cells.

[0146] The MN 20-19 protein was purified from the conditioned media byimmunoaffinity chromatography. 6.5 mg of Mab M75 was coupled to 1 g ofTresyl activated Toyopearl™ [Tosoh, Japan (#14471)]. Approximately 150ml of the conditioned media was run through the M75-Toyopearl column.The column was washed with PBS, and the MN 20-19 protein was eluted with1.5 M MgCl. The eluted protein was then dialyzed against PBS.

Synthetic and Biologic Production of MN Proteins and Polypeptides

[0147] MN proteins and polypeptides of this invention may be preparednot only by recombinant means but also by synthetic and by otherbiologic means. Synthetic formation of the polypeptide or proteinrequires chemically synthesizing the desired chain of amino acids bymethods well known in the art. Exemplary of other biologic means toprepare the desired polypeptide or protein is to subject to selectiveproteolysis a longer MN polypeptide or protein containing the desiredamino acid sequence; for example, the longer polypeptide or protein canbe split with chemical reagents or with enzymes.

[0148] Chemical synthesis of a peptide is conventional in the art andcan be accomplished, for example, by the Merrifield solid phasesynthesis technique [Merrifield, J., Am. Chem. Soc. 85: 2149-2154(1963); Kent et al., Synthetic Peptides in Biology and Medicine, 29 f.f.eds. Alitalo et al., (Elsevier Science Publishers 1985); and Haug, J.D., “Peptide Synthesis and Protecting Group Strategy”, AmericanBiotechnology Laboratory, 5(1): 40-47 (January/February 1987)].

[0149] Techniques of chemical peptide synthesis include using automaticpeptide synthesizers employing commercially available protected aminoacids, for example, Biosearch [San Rafael, Calif. (USA)] Models 9500 and9600; Applied Biosystems, Inc. [Foster City, Calif. (USA)] Model 430;Milligen [a division of Millipore Corp.; Bedford, Mass. (USA)] Model9050; and Du Pont's RAMP (Rapid Automated Multiple Peptide Synthesis)[Du Pont Compass, Wilmington, Del. (USA)].

Regulation of MN Expression and MN Promoter

[0150] MN appears to be a novel regulatory protein that is directlyinvolved in the control of cell proliferation and in cellulartransformation. In HeLa cells, the expression of MN is positivelyregulated by cell density. Its level is increased by persistentinfection with LCMV. In hybrid cells between HeLa and normalfibroblasts, MN expression correlates with tumorigenicity. The fact thatMN is not present in nontumorigenic hybrid cells (CGL1), but isexpressed in a tumorigenic segregant lacking chromosome 11, indicatesthat MN is negatively regulated by a putative suppressor in chromosome11.

[0151] Evidence supporting the regulatory role of MN protein was foundin the generation of stable transfectants of NIH 3T3 cells thatconstitutively express MN protein as described in Example 15. As aconsequence of MN expression, the NIH 3T3 cells acquired featuresassociated with a transformed phenotype: altered morphology, increasedsaturation density, proliferative advantage in serum-reduced media,enhanced DNA synthesis and capacity for anchorage-independent growth.Further, as shown in Example 16, flow cytometric analyses ofasynchronous cell populations indicated that the expression of MNprotein leads to accelerated progression of cells through G1 phase,reduction of cell size and the loss of capacity for growth arrest underinappropriate conditions. Also, Example 16 shows that MN expressingcells display a decreased sensitivity to the DNA damaging drug-mitomycinC.

[0152] Nontumorigenic human cells, CGL1 cells, were also transfectedwith the full-length MN cDNA. The same pSG5C-MN construct in combinationwith pSV2neo plasmid as used to transfect the NIH 3T3 cells (Example 15)was used. Also the protocol was the same except that the G418concentration was increased to 1000 μg/ml.

[0153] Out of 15 MN-positive clones (tested by SP-RIA and Westernblotting), 3 were chosen for further analysis. Two MN-negative clonesisolated from CGL1 cells transfected with empty plasmid were added ascontrols. Initial analysis indicates that the morphology and growthhabits of MN-transfected CGL1 cells are not changed dramatically, buttheir proliferation rate and plating efficiency is increased.

[0154] MN cDNA and promoter. When the promoter region from the MNgenomic clone, isolated as described above, was linked to MN cDNA andtransfected into CGL1 hybrid cells, expression of MN protein wasdetectable immediately after selection. However, then it graduallyceased, indicating thus an action of a feedback regulator. The putativeregulatory element appeared to be acting via the MN promoter, becausewhen the full-length cDNA (not containing the promoter) was used fortransfection, no similar effect was observed.

[0155] An “antisense” MN cDNA/MN promoter construct was used totransfect CGL3 cells. The effect was the opposite of that of the CGL1cells transfected with the “sense” construct. Whereas the transfectedCGL1 cells formed colonies several times larger than the control CGL1,the transfected CGL3 cells formed colonies much smaller than the controlCGL3 cells.

[0156] For those experiments, the part of the promoter region that waslinked to the MN cDNA through BamHI site was derived from NcoI-BamHIfragment of the MN genomic clone and represents the region 233 bpupstream from the transcription initiation site. After the ligation, thejoint DNA was inserted into a pBK-CMV expression vector [Stratagene].The required orientation of the inserted sequence was ensured bydirectional cloning and subsequently verified by restriction analysis.The tranfection procedure was the same as used in transfecting the NIH3T3 cells (Example 15), but co-transfection with the pSV2neo plasmid wasnot necessary since the neo selection marker was already included in thepBK-CMV vector.

[0157] After two weeks of selection in a medium containing G418,remarkable differences between the numbers and sizes of the coloniesgrown were evident as noted above. Immediately following the selectionand cloning, the MN-transfected CGL1 and CGL3 cells were tested bySP-RIA for expression and repression of MN, respectively. The isolatedtransfected CGL1 clones were MN positive (although the level was lowerthan obtained with the full-length cDNA), whereas MN protein was almostabsent from the transfected CGL3 clones. However, in subsequentpassages, the expression of MN in transfected CGL1 cells started tocease, and was then blocked perhaps evidencing a control feedbackmechanism.

[0158] As a result of the very much lowered proliferation of thetransfected CGL3 cells, it was difficult to expand the majority ofcloned cells (according to SP-RIA, those with the lowest levels of MN),and they were lost during passaging. However, some clones overcame thatproblem and again expressed MN. It is possible that once those cellsreached a higher quantity, that the level of endogenously produced MNmRNA increased over the amount of ectopically expressed antisense mRNA.

Nucleic Acid Probes and Test Kits

[0159] Nucleic acid probes of this invention are those comprisingsequences that are complementary or substantially complementary to theMN cDNA sequences shown in FIGS. 1A-1B and 15 or to other MN genesequences, such as, the genomic clone sequence of FIG. 25 [SEQ. ID. NO.:23]. The phrase “substantially complementary” is defined herein to havethe meaning as it is well understood in the art and, thus, used in thecontext of standard hybridization conditions. The stringency ofhybridization conditions can be adjusted to control the precision ofcomplementarity. Exemplary are the stringent hybridization conditionsused in Examples 11 and 12. Two nucleic acids are, for example,substantially complementary to each other, if they hybridize to eachother under such stringent hybridization conditions.

[0160] Stringent hybridization conditions are considered herein toconform to standard hybridization conditions understood in the art to bestringent. For example, it is generally understood that stringentconditions encompass relatively low salt and/or high temperatureconditions, such as provided by 0.02 M to 0.15 M NaCl at temperatures of50C to 70° C. Less stringent conditions, such as, 0.15 M to 0.9 M saltat temperatures ranging from 20° C. to 55° C. can be made more stringentby adding increasing amounts of formamide, which serves to destabilizehybrid duplexes as does increased temperature.

[0161] Exemplary stringent hybridization conditions are described inExamples 11 and 12, infra; the hybridizations therein were carried out“in the presence of 50% formamide at 42° C.” [See Sambrook et al.,Molecular Cloning: A Laboratory Manual, pages 1.91 and 9.47-9.51 (SecondEdition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.;1989); Maniatis et al., Molecular Cloning: A Laboratory Manual, pages387-389 (Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.; 1982);Tsuchiya et al., Oral Surgery. Oral Medicine, Oral Pathology, 71(6):721-725 (June 1991).]

[0162] Preferred nucleic acid probes of this invention are fragments ofthe isolated nucleic acid sequences that encode MN proteins orpolypeptides according to this invention. Preferably those probes arecomposed of at least fifty nucleotides.

[0163] However, nucleic acid probes of this invention need not hybridizeto a coding region of MN. For example, nucleic acid probes of thisinvention may hybridize partially or wholly to a non-coding region ofthe genomic clone of FIG. 25a-b [SEQ. ID. NO.: 23]. Conventionaltechnology can be used to determine whether fragments of SEQ. ID. NO.:23 or related nucleic acids are useful to identify MN nucleic acidsequences. [See, for example, Benton and Davis, supra and Fuscoe et al.,supra.]

[0164] Nucleic acid probes of this invention can be used to detect MNDNA and/or RNA, and thus can be used to test for the presence or absenceof MN genes, and amplification(s), mutation(s) or genetic rearrangementsof MN genes in the cells of a patient. For example, overexpression of anMN gene may be detected by Northern blotting using probes of thisinvention. Gene alterations, as amplifications, translocations,inversions, and deletions among others, can be detected by using probesof this invention for in situ hybridization to chromosomes from apatient's cells, whether in metaphase spreads or interphase nuclei.Southern blotting could also be used with the probes of this inventionto detect amplifications or deletions of MN genes. Restriction FragmentLength Polymorphism (RFLP) analysis using said probes is a preferredmethod of detecting gene alterations, mutations and deletions. Saidprobes can also be used to identify MN proteins and/or polypeptides aswell as homologs or near homologs thereto by their hybridization tovarious mRNAs transcribed from MN genes in different tissues.

[0165] Probes of this invention thus can be usefuldiagnostically/prognostically. Said probes can be embodied in test kits,preferably with appropriate means to enable said probes when hybridizedto an appropriate MN gene or MN mRNA target to be visualized. Suchsamples include tissue specimens including smears, body fluids andtissue and cell extracts.

[0166] PCR Assays. To detect relatively large genetic rearrangements,hybridization tests can be used. To detect relatively small geneticrearrangements, as, for example, small deletions or amplifications, orpoint mutations, the polymerase chain reaction (PCR) would preferably beused. [U.S. Pat. Nos. 4,800,159; 4,683,195; 4,683,202; and Chapter 14 ofSambrook et al., Molecular Cloning: A Laboratory Manual, supra]

[0167] An exemplary assay would use cellular DNA from normal andcancerous cells, which DNA would be isolated and amplified employingappropriate PCR primers. The PCR products would be compared, preferablyinitially, on a sizing gel to detect size changes indicative of certaingenetic rearrangements. If no differences in sizes are noted, furthercomparisons can be made, preferably using, for example,PCR-single-strand conformation polymorphism (PCR-SSCP) assay or adenaturing gradient gel electrophoretic assay. [See, for example,Hayashi, K., “PCR-SSCP: A Simple and Sensitive Method for Detection ofMutations in the Genomic DNA,” in PCR Methods and Applications, 1: 34-38(1991); and Meyers et al., “Detection and Localization of Single BaseChanges by Denaturing Gradient Gel Electrophoresis,” Methods inEnzymology, 155: 501 (1987).]

Assays

[0168] Assays according to this invention are provided to detect and/orquantitate MN antigen or MN-specific antibodies in vertebrate samples,preferably mammalian samples, more preferably human samples. Suchsamples include tissue specimens, body fluids, tissue extracts and cellextracts. MN antigen may be detected by immunoassay, immunohistochemicalstaining, immunoelectron and scanning microscopy using immunogold amongother techniques.

[0169] Preferred tissue specimens to assay by immunohistochemicalstaining include cell smears, histological sections from biopsiedtissues or organs, and imprint preparations among other tissue samples.Such tissue specimens can be variously maintained, for example, they canbe fresh, frozen, or formalin-, alcohol- or acetone- or otherwise fixedand/or paraffin-embedded and deparaffinized. Biopsied tissue samples canbe, for example, those samples removed by aspiration, bite, brush, cone,chorionic villus, endoscopic, excisional, incisional, needle,percutaneous punch, and surface biopsies, among other biopsy techniques.

[0170] Preferred cervical tissue specimens include cervical smears,conization specimens, histologic sections from hysterectomy specimens orother biopsied cervical tissue samples. Preferred means of obtainingcervical smears include routine swab, scraping or cytobrush techniques,among other means. More preferred are cytobrush or swab techniques.Preferably, cell smears are made on microscope slides, fixed, forexample, with 55% EtOH or an alcohol based spray fixative and air-dried.

[0171] Papanicolaou-stained cervical smears (Pap smears) can be screenedby the methods of this invention, for example, for retrospectivestudies. Preferably, Pap smears-would be decolorized and re-stained withlabeled antibodies against MN antigen. Also archival specimens, forexample, matched smears and biopsy and/or tumor specimens, can be usedfor retrospective studies. Prospective studies can also be done withmatched specimens from patients that have a higher than normal risk ofexhibiting abnormal cervical cytopathology.

[0172] Preferred samples in which to assay MN antigen by, for example,Western blotting or radioimmunoassay, are tissue and/or cell extracts.However, MN antigen may be detected in body fluids, which can includeamong other fluids: blood, serum, plasma, semen, breast exudate, saliva,tears, sputum, mucous, urine, lymph, cytosols, ascites, pleuraleffusions, amniotic fluid, bladder washes, bronchioalveolar lavages andcerebrospinal fluid. It is preferred that the MN antigen be concentratedfrom a larger volume of body fluid before testing. Preferred body fluidsto assay would depend on the type of cancer for which one was testing,but in general preferred body fluids would be breast exudate, pleuraleffusions and ascites.

[0173] MN-specific antibodies can be bound by serologically active MNproteins/polypeptides in samples of such body fluids as blood, plasma,serum, lymph, mucous, tears, urine, spinal fluid and saliva; however,such antibodies are found most usually in blood, plasma and serum,preferably in serum. A representative assay to detect MN-specificantibodies is shown in Example 8 below wherein the fusion proteinGEX-3X-MN is used. Correlation of the results from the assays to detectand/or quantitate MN antigen and MN-specific antibodies reactivetherewith, provides a preferred profile of the disease condition of apatient.

[0174] The assays of this invention are both diagnostic and/orprognostic, i.e., diagnostic/prognostic. The term“diagnostic/prognostic” is herein defined to encompass the followingprocesses either individually or cumulatively depending upon theclinical context: determining the presence of disease, determining thenature of a disease, distinguishing one disease from another,forecasting as to the probable outcome of a disease state, determiningthe prospect as to recovery from a disease as indicated by the natureand symptoms of a case, monitoring the disease status of a patient,monitoring a patient for recurrence of disease, and/or determining thepreferred therapeutic regimen for a patient. The diagnostic/prognosticmethods of this invention are useful, for example, for screeningpopulations for the presence of neoplastic or pre-neoplastic disease,determining the risk of developing neoplastic disease, diagnosing thepresence of neoplastic and/or pre-neoplastic disease, monitoring thedisease status of patients with neoplastic disease, and/or determiningthe prognosis for the course of neoplastic disease. For example, itappears that the intensity of the immunostaining with MN-specificantibodies may correlate with the severity of dysplasia present insamples tested.

[0175] The present invention is useful for screening for the presence ofa wide variety of neoplastic diseases including carcinomas, such as,mammary, urinary tract, ovarian, uterine, cervical, endometrial,squamous cell and adenosquamous carcinomas; head and neck cancers;mesodermal tumors, such as, neuroblastomas and retinoblastomas;sarcomas, such as osteosarcomas and Ewing's sarcoma; and melanomas. Ofparticular interest are gynecological cancers including ovarian,uterine, cervical, vaginal, vulval and endometrial cancers, particularlyovarian, uterine cervical and endometrial cancers. Also of particularinterest are cancers of the breast, of the stomach including esophagus,of the colon, of the kidney, of the prostate, of the liver, of theurinary tract including bladder, of the lung, and of the head and neck.

[0176] The invention provides methods and compositions for evaluatingthe probability of the presence of malignant or pre-malignant cells, forexample, in a group of cells freshly removed from a host. Such an assaycan be used to detect tumors, quantitate their growth, and help in thediagnosis and prognosis of disease. The assays can also be used todetect the presence of cancer metastasis, as well as confirm the absenceor removal of all tumor tissue following surgery, cancer chemotherapyand/or radiation therapy. It can further be used to monitor cancerchemotherapy and tumor reappearance.

[0177] The presence of MN antigen or antibodies can be detected and/orquantitated using a number of well-defined diagnostic assays. Those inthe art can adapt any of the conventional immunoassay formats to detectand/or quantitate MN antigen and/or antibodies. Example 8 details theformat of a preferred diagnostic method of this invention—aradioimmunoassay. Immunohistochemical staining is another preferredassay format as exemplified in Example 13.

[0178] Many other formats for detection of MN antigen and MN-specificantibodies are, of course available. Those can be Western blots, ELISAs(enzyme-linked immunosorbent assays), RIAs (radioimmunoassay),competitive EIA or dual antibody sandwich assays, among other assays allcommonly used in the diagnostic industry. In such immunoassays, theinterpretation of the results is based on the assumption that theantibody or antibody combination will not cross-react with otherproteins and protein fragments present in the sample that are unrelatedto MN.

[0179] Representative of one type of ELISA test for MN antigen is aformat wherein a microtiter plate is coated with antibodies made to MNproteins/polypeptides or antibodies made to whole cells expressing MNproteins, and to this is added a patient sample, for example, a tissueor cell extract. After a period of incubation permitting any antigen tobind to the antibodies, the plate is washed and another set of anti-MNantibodies which are linked to an enzyme is added, incubated to allowreaction to take place, and the plate is then rewashed. Thereafter,enzyme substrate is added to the microtiter plate and incubated for aperiod of time to allow the enzyme to work on the substrate, and theadsorbance of the final preparation is measured. A large change inabsorbance indicates a positive result.

[0180] It is also apparent to one skilled in the art of immunoassaysthat MN proteins and/or polypeptides can be used to detect and/orquantitate the presence of MN antigen in the body fluids, tissues and/orcells of patients. In one such embodiment, a competition immunoassay isused, wherein the MN protein/polypeptide is labeled and a body fluid isadded to compete the binding of the labeled MN protein/polypeptide toantibodies specific to MN protein/polypeptide. Such an assay can be usedto detect and/or quantitate MN antigen as described in Example 8.

[0181] In another embodiment, an immunometric assay may be used whereina labeled antibody made to a MN protein or polypeptide is used. In suchan assay, the amount of labeled antibody which complexes with theantigen-bound antibody is directly proportional to the amount of MNantigen in the sample.

[0182] A representative assay to detect MN-specific antibodies is acompetition assay in which labeled MN protein/polypeptide isprecipitated by antibodies in a sample, for example, in combination withmonoclonal antibodies recognizing MN proteins/polypeptides. One skilledin the art could adapt any of the conventional immunoassay formats todetect and/or quantitate MN-specific, antibodies. Detection of thebinding of said antibodies to said MN protein/polypeptide could be bymany ways known to those in the art, e.g., in humans with the use ofanti-human labeled IgG.

[0183] An exemplary immunoassay method of this invention to detectand/or quantitate MN antigen in a vertebrate sample comprises the stepsof:

[0184] a) incubating said vertebrate sample with one or more sets ofantibodies (an antibody or antibodies) that bind to MN antigen whereinone set is labeled or otherwise detectable;

[0185] b) examining the incubated sample for the presence of immunecomplexes comprising MN antigen and said antibodies.

[0186] Another exemplary immunoassay method according to this inventionis that wherein a competition immunoassay is used to detect and/orquantitate MN antigen in a vertebrate sample and wherein said methodcomprises the steps of:

[0187] a) incubating a vertebrate sample with one or more sets ofMN-specific antibodies and a certain amount of a labeled or otherwisedetectable MN protein/polypeptide wherein said MN protein/polypeptidecompetes for binding to said antibodies with MN antigen present in thesample;

[0188] b) examining the incubated sample to determine the amount oflabeled/detectable MN protein/polypeptide bound to said antibodies; and

[0189] c) determining from the results of the examination in step b)whether MN antigen is present in said sample and/or the amount of MNantigen present in said sample.

[0190] Once antibodies (including biologically active antibodyfragments) having suitable specificity have been prepared, a widevariety of immunological assay methods are available for determining theformation of specific antibody-antigen complexes. Numerous competitiveand non-competitive protein binding assays have been described in thescientific and patent literature, and a large number of such assays arecommercially available. Exemplary immunoassays which are suitable fordetecting a serum antigen include those described in U.S. Pat. Nos.3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876.

[0191] Antibodies employed in assays may be labeled or unlabeled.Unlabeled antibodies may be employed in agglutination; labeledantibodies may be employed in a wide variety of assays, employing a widevariety of labels.

[0192] Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors, freeradicals, particles, dyes and the like. Such labeled reagents may beused in a variety of well known assays, such as radioimmunoassays,enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and thelike. See for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837;and 4,233,402.

[0193] Methods to prepare antibodies useful in the assays of theinvention are described below. The examples below detail representativeassays according to this invention.

[0194] Immunoassay Test Kits

[0195] The above outlined assays can be embodied in test kits to detectand/or quantitate MN antigen and/or MN-specific antibodies (includingbiologically active antibody fragments). Kits to detect and/orquantitate MN antigen can comprise MN protein(s)/polypeptides(s) and/orMN-specific antibodies, polyclonal and/or monoclonal. Suchdiagnostic/prognostic test kits can comprise one or more sets ofantibodies, polyclonal and/or monoclonal, for a sandwich format whereinantibodies recognize epitopes on the MN antigen, and one set isappropriately labeled or is otherwise detectable.

[0196] Test kits for an assay format wherein there is competitionbetween a labeled (or otherwise detectable) MN protein/polypeptide andMN antigen in the sample, for binding to an antibody, can comprise thecombination of the labeled protein/polypeptide and the antibody inamounts which provide for optimum sensitivity and accuracy.

[0197] Test kits for MN-specific antibodies preferably compriselabeled/detectable MN proteins(s) and/or polypeptides(s), and maycomprise other components as necessary, for example, to perform apreferred assay as outlined in Example 8 below, such as, controls,buffers, diluents and detergents. Such test kits can have otherappropriate formats for conventional assays.

[0198] A kit for use in an enzyme-immunoassay typically includes anenzyme-labelled reagent and a substrate for the enzyme. The enzyme can,for example, bind either an MN-specific antibody of this invention or toan antibody to such an MN-specific antibody.

[0199] Preparation of MN-Specific Antibodies

[0200] The term “antibodies” is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Suchantibodies may be prepared by conventional methodology and/or by geneticengineering. Antibody fragments may be genetically engineered,preferably from the variable regions of the light and/or heavy chains(V_(H) and V_(L)), including the hypervariable regions, and still morepreferably from both the V_(H) and V_(L) regions. For example, the term“antibodies” as used herein comprehends polyclonal and monoclonalantibodies and biologically active fragments thereof including amongother possibilities “univalent” antibodies [Glennie et al., Nature. 295:712 (1982)]; Fab proteins including Fab′ and F(ab′)₂ fragments whethercovalently or non-covalently aggregated; light or heavy chains alone,preferably variable heavy and light chain regions (V_(H) and V_(L)regions), and more preferably including the hypervariable regions[otherwise known as the complementarity determining regions (CDRS) ofsaid V_(H) and V_(L) regions]; F_(c) proteins; “hybrid” antibodiescapable of binding more than one antigen; constant-variable regionchimeras; “composite” immunoglobulins with heavy and light chains ofdifferent origins; “altered” antibodies with improved specificity andother characteristics as prepared by standard recombinant techniques andalso by oligonucleotide-directed mutagenesis techniques[Dalbadie-McFarland et al., PNAS (USA), 79: 6409 (1982)].

[0201] It may be preferred for therapeutic and/or imaging uses that theantibodies be biologically active antibody fragments, preferablygenetically engineered fragments, more preferably genetically engineeredfragments from the V_(H) and/or V₁ regions, and still more preferablycomprising the hypervariable regions thereof.

[0202] There are conventional techniques for making polyclonal andmonoclonal antibodies well-known in the immunoassay art. Immunogens toprepare MN-specific antibodies include MN proteins and/or polypeptides,preferably purified, and MX-infected tumor line cells, for example,MX-infected HeLa cells, among other immunogens.

[0203] Anti-peptide antibodies are also made by conventional methods inthe art as described in European Patent Publication No. 44,710(published Jan. 27, 1982). Briefly, such anti-peptide antibodies areprepared by selecting a peptide from an MN amino acid sequence as fromFIGS. 1A-B or 15, chemically synthesizing it, conjugating it to anappropriate immunogenic protein and injecting it into an appropriateanimal, usually a rabbit or a mouse; then, either polyclonal ormonoclonal antibodies are made, the latter by a Kohler-Milsteinprocedure, for example.

[0204] Besides conventional hybridoma technology, newer technologies canbe used to produce antibodies according to this invention. For example,the use of the PCR to clone and express antibody V-genes and phagedisplay technology to select antibody genes encoding fragments withbinding activities has resulted in the isolation of antibody fragmentsfrom repertoires of PCR amplified V-genes using immunized mice orhumans. [Marks et al., BioTechnology, 10: 779 (July 1992) forreferences; Chiang et al., BioTechniques. 7(4): 360 (1989); Ward et al.,Nature. 341: 544 (Oct. 12, 1989); Marks et al., J. Mol. Biol. 222: 581(1991); Clackson et al., Nature. 352: (15 Aug. 1991); and Mullinax etal., PNAS (USA), 87: 8095 (October 1990).]

[0205] Descriptions of preparing antibodies, which term is hereindefined to include biologically active antibody fragments, byrecombinant techniques can be found in U.S. Pat. No. 4,816,567 (issuedMar. 28, 1989); European Patent Application Publication Number (EP)338,745 (published Oct. 25, 1989); EP 368,684 (published Jun. 16, 1990);EP 239,400 (published Sep. 30, 1987); WO 90/14424 (published Nov. 29,1990); WO 90/14430 (published May 16, 1990); Huse et al., Science. 246:1275 (Dec. 8, 1989); Marks et al., BioTechnology, 10: 779 (July 1992);La Sastry et al., PNAS (USA), 86: 5728 (August 1989); Chiang et al.,BioTechniques, 7(40): 360 (1989); Orlandi et al., PNAS (USA), 86: 3833(May 1989); Ward et al. Nature. 341: 544 (Oct. 12, 1989); Marks et al.,J. Mol. Biol. 222: 581 (1991); and Hoogenboom et al., Nucleic AcidsRes., 19(15): 4133 (1991).

[0206] Representative Mabs

[0207] Monoclonal antibodies for use in the assays of this invention maybe obtained by methods well known in the art for example, Galfre andMilstein, “Preparation of Monoclonal Antibodies: Strategies andProcedures,” in Methods in Enzymology: Immunochemical Techniques, 73:1-46 [Langone and Vanatis (eds); Academic Press (1981)]; and in theclassic reference, Milstein and Kohler, Nature, 256: 495-497 (1975).]

[0208] Although representative hybridomas of this invention are formedby the fusion of murine cell lines, human/human hybridomas [Olsson etal., PNAS (USA), 77: 5429 (1980)] and human/murine hybridomas [Schlom etal., PNAS (USA), 77: 6841 (1980); Shearman et al. J. Immunol., 146:928-935 (1991); and Gorman et al., PNAS (USA), 88: 4181-4185 (1991)] canalso be prepared among other possiblities. Such humanized monoclonalantibodies would be preferred monoclonal antibodies for therapeutic andimaging uses.

[0209] Monoclonal antibodies specific for this invention can be preparedby immunizing appropriate mammals, preferably rodents, more preferablyrabbits or mice, with an appropriate immunogen, for example,MaTu-infected HeLa cells, MN fusion proteins, or MNproteins/polypeptides attached to a carrier protein if necessary.Exemplary methods of producing antibodies of this invention aredescribed below.

[0210] The monoclonal antibodies useful according to this invention toidentify MN proteins/polypeptides can be labeled in any conventionalmanner, for example, with enzymes such as horseradish peroxidase (HRP),fluorescent compounds, or with radioactive isotopes such as, ¹²⁵I, amongother labels. A preferred label, according to this invention is ¹²⁵I,and a preferred method of labeling the antibodies is by usingchloramine-T [Hunter, W. M., “Radioimmunoassay,” In: Handbook ofExperimental Immunology, pp. 14.1-14.40 (D W. Weir ed.; Blackwell,oxford/London/Edinburgh/Melbourne; 1978)].

[0211] Representative mabs of this invention include Mabs M75, MN9, MN12and MN7 described below. Monoclonal antibodies of this invention serveto identify MN proteins/polypeptides in various laboratory diagnostictests, for example, in tumor cell cultures or in clinical samples.

[0212] Mabs Prepared Against HeLa Cells

[0213] MAb M75. Monoclonal antibody M75 (MAb M75) is produced by mouselymphocytic hybridoma VU-M75, which was initially deposited in theCollection of Hybridomas at the Institute of Virology, Slovak Academy ofSciences (Bratislava, Czechoslovakia) and was deposited under ATCCDesignation HB 11128 on Sep. 17, 1992 at the American Type CultureCollection (ATCC) in Rockville, Md. (USA).

[0214] Hybridoma VU-M75 was produced according to the proceduredescribed in Gerhard, W., “Fusion of cells in suspension and outgrowthof hybrids in conditioned medium,” In: Monoclonal Antibodies.Hybridomas: A New Dimension in Biological Analysis, page 370 [Kennet etal. (eds.); Plenum N.Y. (USA)]. BALB/C mice were immunized withMaTu-infected HeLa cells, and their spleen cells were fused with myelomacell line NS-0. Tissue culture media from the hybridomas were screenedfor monoclonal antibodies, using as antigen the p58 immunoprecipitatedfrom cell extracts of MaTu-infected HeLa with rabbit anti-MaTu serum andprotein A-Staphylococcus aureus cells (SAC) [Zavada and Zavadova, Arch.Virol. 118 189-197 (1991)], and eluted from SDS-PAGE gels. Monoclonalantibodies were purified from TC media by affinity chromatography onprotein A-Sepharose [Harlow and Lane, “Antibodies: A Laboratory Manual,”Cold Spring Harbor, Cold Spring Harbor, N.Y. (USA); 1988].

[0215] Mab M75 recognizes both the nonglycosylated GEX-3X-MN fusionprotein and native MN protein as expressed in CGL3 cells equally well.Mab M75 was shown by epitope mapping to be reactive with the epitoperepresented by the amino acid sequence from AA 62 to AA 67 [SEQ. ID.NO.: 10] of the MN protein shown in FIG. 15.

[0216] Mabs M16 and M67. Also produced by the method described forproducing MAb M75 (isotype IgG2B) were MAbs M16 (isotype IgG2A) and M67(isotype IgG1). Mabs M16 and M67 recognize MX protein, as described inthe examples below.

[0217] MAb H460. Monoclonal antibody H460 (MAb H460) was prepared in amanner similar to that for MAb M75 except that the mice were immunizedwith HeLa cells uninfected with MaTu, and lymphocytes of the mice ratherthan spleen cells were fused with cells from myeloma cell line NS-0. MAbH460 reacts about equally with any human cells.

[0218] Mabs Prepared Against Fusion Protein GEX-3X-MN

[0219] Monoclonal antibodies of this invention were also preparedagainst the MN glutathione S-transferase fusion protein (GEX-3X-MN)purified by affinity chromatography as described above. BALB/C mice wereimmunized intraperitoneally according to standard procedures with theGEX-3X-MN fusion protein in Freund's adjuvant. Spleen cells of the micewere fused with SP/20 myeloma cells [Milstein and Kohler, supra].

[0220] Tissue culture media from the hybridomas were screened againstCGL3 and CGL1 membrane extracts in an ELISA employing HRPlabelled-rabbit anti-mouse. The membrane extracts were coated ontomicrotiter plates. Selected were antibodies reacted with the CGL3membrane extract. Selected hybridomas were cloned twice by limitingdilution.

[0221] The mabs prepared by the just described method were characterizedby Western blots of the GEX-3X-MN fusion protein, and with membraneextracts from the CGL1 and CGL3 cells. Representative of the mabsprepared are Mabs MN9, MN12 and MN7.

[0222] Mab MN9. Monoclonal antibody MN9 (Mab MN9) reacts to the sameepitope as Mab M75, represented by the sequence from AA 62 to AA 67[SEQ. ID. NO.: 10] of the FIG. 15 MN protein. As Mab M75, Mab MN9recognizes both the GEX-3X-MN fusion protein and native MN proteinequally well.

[0223] Mabs corresponding to Mab MN9 can be prepared reproducibly byscreening a series of mabs prepared against an MN protein/polypeptide,such as, the GEX-3X-MN fusion protein, against the peptide representingthe epitope for Mabs M75 and MN9. That peptide is Arg Arg Ile Cys ProVal [SEQ. ID. NO.: 10]. Alternatively, the Novatope system [Novagen] orcompetition with the deposited Mab M75 could be used to select mabscomparable to Mabs M75 and MN9.

[0224] Mab MN12. Monoclonal antibody MN12 (Mab MN12) is produced by themouse lymphocytic hybridoma MN 12.2.2 which was deposited under ATCCDesignation HB 11647 on Jun. 9, 1994 at the American Type CultureCollection (ATCC) at 12301 Parklawn Drive, Rockville, Md. 20852 (USA).Antibodies corresponding to Mab MN12 can also be made, analogously tothe method outlined above for Mab MN9, by screening a series ofantibodies prepared against an MN protein/polypeptide, against thepeptide representing the epitope for Mab MN12. That peptide is Gly LysMet Thr His Trp [SEQ. ID. NO.: 11]. The Novatope system could also beused to find antibodies specific for said pepitope.

[0225] Mab MN7. Monoclonal antibody MN7 (Mab MN7) was selected from mabsprepared against nonglycosylated GEX-3X-MN as described above. Itrecognizes the epitope on MN represented by the amino acid sequence fromAA 127 to AA 147 [SEQ. ID. NO.: 12; Asn Asn Ala His Arg Asp Lys Glu GlyAsp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro] of the FIG. 15 MNprotein. Analogously to methods described above for Mabs MN9 and MN12,mabs corresponding to Mab MN7 can be prepared by selecting mabs preparedagainst an MN protein/polypeptide that are reactive with the peptidehaving SEQ. ID. NO.: 12, or by the stated alternative means.

[0226] Epitope Mapping

[0227] Epitope mapping was performed by the Novatope system, a kit forwhich is commercially available from Novagen, Inc. [See, for analogousexample, Li et al., Nature. 363: 85-88 (6 May 1993).] In brief, the MNcDNA was cut into overlapping short fragments of approximately 60 basepairs. The fragments were expressed in E. coli, and the E. coli colonieswere transferred onto nitrocellulose paper, lysed and probed with themab of interest. The MN cDNA of clones reactive with the mab of interestwas sequenced, and the epitopes of the mabs were deduced from theoverlapping polypeptides found to be reactive with each mab.

[0228] Therapeutic Use of MN-Specific Antibodies

[0229] The MN-specific antibodies of this invention, monoclonal and/orpolyclonal, preferably monoclonal, and as outlined above, may be usedtherapeutically in the treatment of neoplastic and/or pre-neoplasticdisease, either alone or in combination with chemotherapeutic drugs ortoxic agents, such as ricin A. Further preferred for therapeutic usewould be biologically active antibody fragments as described herein.Also preferred MN-specific antibodies for such therapeutic uses would behumanized monoclonal antibodies.

[0230] The MN-specific antibodies can be administered in atherapeutically effective amount, preferably dispersed in aphysiologically acceptable, nontoxic liquid vehicle.

[0231] Imaging Use of Antibodies

[0232] Further, the MN-specific antibodies of this invention when linkedto an imaging agent, such as a radionuclide, can be used for imaging.Biologically active antibody fragments or humanized monoclonalantibodies, may be preferred for imaging use.

[0233] A patient's neoplastic tissue can be identified as, for example,sites of transformed stem cells, of tumors and locations of anymetastases. Antibodies, appropriately labeled or linked to an imagingagent, can be injected in a physiologically acceptable carrier into apatient, and the binding of the antibodies can be detected by a methodappropriate to the label or imaging agent, for example, by scintigraphy.

Antisense MN Nucleic Acid Sequences

[0234] Antisense MN Nucleic Acid Sequences MN genes are hereinconsidered putative oncogenes and the encoded proteins thereby areconsidered to be putative oncoproteins. Antisense nucleic acid sequencessubstantially complementary to mRNA transcribed from MN genes, asrepresented by the antisense oligodeoxynucleotides (ODNs) of Example 10,infra, can be used to reduce or prevent expression of the MN gene.[Zamecnick, P C., “Introduction: Oligonucleotide Base Hybridization as aModulator of Genetic Message Readout,” pp. 1-6, Prospects for AntisenseNucleic Acid Therapy of Cancer and AIDS, (Wiley-Liss, Inc., New York,N.Y., USA; 1991); Wickstrom, E., “Antisense DNA Treatment of HL-60Promyelocytic Leukemia Cells: Terminal Differentiation and Dependence onTarget Sequence,” pp. 7-24, id.; Leserman et al., “Targeting andIntracellular Delivery of Antisense Oligonucleotides Interfering withOncogene Expression,” pp. 25-34, id.; Yokoyama, K., “TranscriptionalRegulation of c-myc Proto-oncogene by Antisense RNA,” pp. 35-52, id.;van den Berg et al., “Antisense fos oligodeoxyribonucleotides Suppressthe Generation of Chromosomal Aberrations,” pp. 63-70, id.; Mercola, D.,“Antisense fos and fun RNA,” pp. 83-114, id.; Inouye, Gene. 72: 25-34(1988); Miller and Ts'o, Ann. Reports Med. Chem., 23: 295-304 (1988);Stein and Cohen, Cancer Res. 48: 2659-2668 (1988); Stevenson andInversen, J. Gen. Virol., 70: 2673-2682 (1989); Goodchild, “Inhibitionof Gene Expression by oligonucleotides,” pp. 53-77,Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression (Cohen, JS., ed; CRC Press, Boca Raton, Fla., USA; 1989); Dervan et al.,“Oligonucleotide Recognition of Double-helical DNA by Triple-helixFormation,” pp. 197-210, id.; Neckers, L M., “AntisenseOligodeoxynucleotides as a Tool for Studying Cell Regulation: Mechanismsof Uptake and Application to the Study of Oncogene Function,” pp.211-232, id.; Leitner et al., PNAS (USA), 87: 3430-3434 (1990);Bevilacqua et al., PNAS (USA), 85: 831-835 (1988); Loke et al. Curr.Top. Microbiol. Immunol. 141: 282-288 (1988); Sarin et al., PNAS (USA),85: 7448-7451 (1988); Agrawal et al., “Antisense Oligonucleotides: APossible Approach for Chemotherapy and AIDS,” International Union ofBiochemistry Conference on Nucleic Acid Therapeutics (Jan. 13-17, 1991;Clearwater Beach, Fla., USA); Armstrong, L., Ber. Week, pp. 88-89 (Mar.5, 1990); and Weintraub et al., Trends. 1: 22-25 (1985).] Such antisensenucleic acid sequences, preferably oligonucleotides, by hybridizing tothe MN mRNA, particularly in the vicinity of the ribosome binding siteand translation initiation point, inhibits translation of the mRNA.Thus, the use of such antisense nucleic acid sequences may be consideredto be a form of cancer therapy.

[0235] Preferred antisense oligonucleotides according to this inventionare gene-specific ODNs or oligonucleotides complementary to the 5′ endof MN mRNA. Particularly preferred are the 29-mer ODN1 and 19-mer ODN2for which the sequences are provided in Example 10, infra. Thoseantisense ODNs are representative of the many antisense nucleic acidsequences that can function to inhibit MN gene expression. Ones ofordinary skill in the art could determine appropriate antisense nucleicacid sequences, preferably antisense oligonucleotides, from the nucleicacid sequences of FIGS. 1A-1B, 15 and 25 a-b.

[0236] Also, as described above, CGL3 cells transfected with an“antisense” MN cDNA/promoter construct formed colonies much smaller thancontrol CGL3 cells.

Vaccines

[0237] It will be readily appreciated that MN proteins and polypeptidesof this invention can be incorporated into vaccines capable of inducingprotective immunity against neoplastic disease and a dampening effectupon tumorigenic activity. Efficacy of a representative MN fusionprotein GEX-3X-MN as a vaccine in a rat model is shown in Example 14.

[0238] MN proteins and/or polypeptides may be synthesized or preparedrecombinantly or otherwise biologically, to comprise one or more aminoacid sequences corresponding to one or more epitopes of the MN proteinseither in monomeric or multimeric form. Those proteins and/orpolypeptides may then be incorporated into vaccines capable of inducingprotective immunity. Techniques for enhancing the antigenicity of suchpolypeptides include incorporation into a multimeric structure, bindingto a highly immunogenic protein carrier, for example, keyhole limpethemocyanin (KLH), or diptheria toxoid, and administration in combinationwith adjuvants or any other enhancers of immune response.

[0239] Preferred MN proteins/polypeptides to be used in a vaccineaccording to this invention would be genetically engineered MN proteins.Preferred recombinant MN protein are the GEX-3X-MN and MN 20-19proteins.

[0240] A preferred exemplary use of such a vaccine of this inventionwould be its administration to patients whose MN-carrying primary cancerhad been surgically removed. The vaccine may induce active immunity inthe patients and prevent recidivism or metastasis.

[0241] It will further be appreciated that anti-idiotype antibodies toantibodies to MN proteins/polypeptides are also useful as vaccines andcan be similarly formulated.

[0242] An amino acid sequence corresponding to an epitope of an MNprotein/polypeptide either in monomeric or multimeric form may also beobtained by chemical synthetic means or by purification from biologicalsources including genetically modified microorganisms or their culturemedia. [See Lerner, “Synthetic Vaccines”, Sci. Am. 248(2): 66-74(1983).] The protein/polypeptide may be combined in an amino acidsequence with other proteins/polypeptides including fragments of otherproteins, as for example, when synthesized as a fusion protein, orlinked to other antigenic or non-antigeneic polypeptides of synthetic orbiological origin. In some instances, it may be desirable to fuse a MNprotein or polypeptide to an immunogenic and/or antigenic protein orpolypeptide, for example, to stimulate efficacy of a MN-based vaccine.

[0243] The term “corresponding to an epitope of an MNprotein/polypeptide” will be understood to include the practicalpossibility that, in some instances, amino acid sequence variations of anaturally occurring protein or polypeptide may be antigenic and conferprotective immunity against neoplastic disease and/or anti-tumorigeniceffects. Possible sequence variations include, without limitation, aminoacid substitutions, extensions, deletions, truncations, interpolationsand combinations thereof. Such variations fall within the contemplatedscope of the invention provided the protein or polypeptide containingthem is immunogenic and antibodies elicited by such a polypeptide orprotein cross-react with naturally occurring MN proteins andpolypeptides to a sufficient extent to provide protective immunityand/or anti-tumorigenic activity when administered as a vaccine.

[0244] Such vaccine compositions will be combined with a physiologicallyacceptable medium, including immunologically acceptable diluents andcarriers as well as commonly employed adjuvants such as Freund'sComplete Adjuvant, saponin, alum, and the like. Administration would bein immunologically effective amounts of the MN proteins or polypeptides,preferably in quantities providing unit doses of from 0.01 to 10.0micrograms of immunologically active MN protein and/or polypeptide perkilogram of the recipient's body weight. Total protective doses mayrange from 0.1 to about 100 micrograms of antigen.

[0245] Routes of administration, antigen dose, number and frequency ofinjections are all matters of optimization within the scope of theordinary skill in the art.

[0246] The following examples are for purposes of illustration only andnot meant to limit the invention in any way.

Materials and Methods

[0247] The following materials and methods were used in examples below.

[0248] MaTu-Infected and Uninfected HeLa Cells

[0249] MaTu agent [Zavada et al., Nature New Biol., 240: 124-125 (1972);Zavada et al., J. Gen. Virol, 24: 327-337 (1974)] was from original“MaTu” cells [Widmaier et al., Arch. Geschwulstforsch, 44: 1-10 (1974)]transferred into our stock of HeLa by cocultivation with MaTu cellstreated with mitomycin C, to ensure that control and MaTu-infected cellswere comparable. MaTu cells were incubated for 3 hours at 37° C. inmedia with 5 μg/ml of mitomycin C [Calbiochem; LaJolla, Calif. (USA)].Mixed cultures were set to 2×10⁵ of mitomycin C-treated cells and 4×10⁵of fresh recipient cells in 5 ml of medium. After 3 days they were firstsubcultured and further passaged 1-2 times weekly.

[0250] Control HeLa cells were the same as those described in Zavada etal. (1972), supra.

[0251] Sera

[0252] Human sera from cancer patients, from patients suffering withvarious non-tumor complaints and from healthy women were obtained fromthe Clinics of Obstetrics and Gynaecology at the Postgraduate MedicalSchool, Bratislava, Czechoslovakia. Human sera KH was from a fifty yearold mammary carcinoma patient, fourteen months after resection. Thatserum was one of two sera out of 401 serum samples that containedneutralizing antibodies to the VSV(MaTU) pseudotype as described inZavada et al. (1972), supra. Serum L8 was from a patient with Paget'sdisease. Serum M7 was from a healthy donor.

[0253] Rabbit anti-MaTu serum was prepared by immunizing a rabbit threetimes at intervals of 30 days with 10-5×10⁷ viable MaTu-infected HeLacells.

[0254] RIP and PAGE

[0255] RIP and PAGE were performed essentially as described in Zavadaand Zavadova, supra, except that in the experiments described herein[³⁵S]methionine (NEN), 10 μCi/ml of methionine-free MEM medium,supplemented with 2% FCS and 3% complete MEM were used. Confluent petridish cultures of cells were incubated overnight in that media.

[0256] For RIP, the SAC procedure [Kessler, J. Immunol., 115: 1617-1624(1975)] was used. All incubations and centrifugations were performed at0-4° C. Cell monolayers were extracted with RIPA buffer (0.14 M NaCl,7.5 mM phosphate buffer, pH 7.2, 1% Triton X-100, 0.1% sodiumdeoxycholate, 1 mM phenylmethylsulfonyl fluoride and Trasylol). Toreduce non-specific reactions, antisera were preabsorbed with fetal calfserum [Barbacid et al., PNAS (USA), 77: 1617-1621 (1980)] and antigenicextracts with SAC.

[0257] For PAGE (under reducing conditions) we used 10% gels with SDS[Laemmli, Nature, 227: 680-685 (1970)]. As reference marker proteinsserved the Sigma kit [product MW-SDS-200; St. Louis, Mo. (USA)]. Forfluorography we used salicylate [Heegaard et al., Electrophoresis, 5:263-269 (1984)].

[0258] Immunoblots

[0259] Immunoblotting used as described herein follows the method ofTowbin et al., PNAS (USA), 76: 4350-4354 (1979). The proteins weretransferred from the gels onto nitrocellulose [Schleicher and Schuell;Dassel Germany; 0.45 μm porosity] in Laemmli electrode buffer diluted1:10 with distilled water, with no methanol or SDS. The transfer was for2½ hours at 1.75 mA/cm². The blots were developed with ¹²⁵I-labeled MAbsand autoradiography was performed using intensifying screens, with X-rayfilms exposed at −70° C.

[0260] In extracts from cell cultures containing only small amounts ofMN antigen, we concentrated the antigen from 0.5 or 1 ml of an extractby adding 50 μl of a 10% SAC suspension, pre-loaded with MAb M75. Thismethod allowed the concentration of MN antigen even from clinicalspecimens, containing human IgG; preliminary control experiments showedthat such a method did not interfere with the binding of the MN antigento SAC-adsorbed M75. Tissue extracts were made by grinding the tissuewith a mortar and pestle and sand (analytical grade). To the homogenateswas added RIPA buffer, 10:1 (volume to weight) of original tissue. Theextracts were clarified for 3 minutes on an Eppendorf centrifuge.

EXAMPLE 1 Immunofluorescence of MaTu-Specific Antigens

[0261] Immunofluorescence experiments were performed on control andMaTu-infected HeLa cells with monoclonal antibodies, prepared asdescribed above, which are specific for MaTu-related antigens.FITC-conjugated anti-mouse IgG was used to detect the presence of themonoclonal antibodies. Staining of the cells with Giemsa revealed noclear differences between control and MaTu-infected HeLa cells.

[0262] MAbs, which in preliminary tests proved to be specific forMaTu-related antigens, showed two different reactivities inimmunofluorescence. A representative of the first group, MAb M67, gave agranular cytoplasmic fluorescence in MaTu-infected HeLa, which was onlyseen in cells fixed with acetone; living cells showed no fluorescence.MAb M16 gave the same type of fluorescence. With either M67 or M16, onlyextremely weak “background” fluorescence was seen in control HeLa cells.

[0263] Another MAb, M75, showed a granular membrane fluorescence onliving MaTu-infected cells and a granular nuclear fluorescence inacetone-fixed cells. However, M75 sometimes showed a similar, althoughmuch weaker, fluorescence on uninfected HeLa cells. A relationship wasobserved based upon the conditions of growth: in HeLa cells uninfectedwith MaTu, both types of fluorescence with MAb M75 were observed only ifthe cells were grown for several passages in dense cultures, but not insparse ones.

[0264] The amount of M75-reactive cell surface antigen was analyzedcytofluorometrically and was dependent on the density of the cellcultures and on infection with MaTu. Control and MaTu infected HeLacells were grown for 12 days in dense or sparse cultures. The cells werereleased with Versene (EDTA), and incubated with MAb M75 or with no MAb,and subsequently incubated with FITC-conjugated anti-mouse IgG. Theintensity of fluorescence was measured.

[0265] It appeared that the antigen binding MAb M75 is inducible: it wasfound to be absent in control HeLa grown in sparse culture, and to beinduced either by the growth of HeLa in dense culture or by infectionwith MaTu. Those two factors were found to have an additive orsynergistic effect. Those observations indicated along with otherresults described herein that there were two different agents involved:exogenous, transmissible MX, reactive with M67, and endogenous,inducible MN, detected by MAb M75.

EXAMPLE 2 Immunoblot Analysis of Protein(s) Reactive with MAb M75

[0266] To determine whether MAb M75 reacts with the same protein in bothuninfected and MaTu-infected HeLa, and to determine the molecular weightof the protein, extracts of those cells were analyzed by PAGE andimmunoblotting (as described above). HeLa cells uninfected orMaTu-infected, that had been grown for 12 days in dense or sparsecultures, were seeded in 5-cm petri dishes, all variants at 5×10⁵ cellsper dish. Two days later, the cells were extracted with RIPA buffer(above described), 200 μl/dish. The extracts were mixed with 2×concentrated Laemmli sample buffer containing 6% mercaptoethanol andboiled for five minutes. Proteins were separated by SDS-PAGE and blottedon nitrocellulose. The blots were developed with ¹²⁵I-labeled MAb M75and autoradiography.

[0267] MAb M75 reacted with two MN-specific protein bands of 54 kd and58 kd, which were the same in uninfected HeLa grown at high density andin MaTu-infected HeLa, evidencing that M75 recognizes the sameprotein(s) in both uninfected and MaTu-infected HeLa cells. Consistentwith the cytofluorometric results, the amount of the antigen dependedboth on cell density and on infection with MaTu, the latter being a muchmore potent inducer of p54/58N.

EXAMPLE 3 Radioimmunoassay of MaTu-Specific Antigens In Situ

[0268] In contrast to the results with M75, the other MAb, M67, appearedto be specific for the exogenous, transmissible agent MX. With M67 weobserved no immunofluorescence in control HeLa, regardless of whetherthe cells were grown in dense or in sparse culture. That difference wasclearly evidenced in radioimmunoassay experiments wherein ¹²⁵I-labeledMAbs M67 and M75 were used.

[0269] For such experiments, parallel cultures of uninfected andMaTu-infected cells were grown in dense or sparse cultures. The cultureswere either live (without fixation), or fixed (with methanol for fiveminutes and air-dried). The cultures were incubated for two hours inpetri dishes with the ¹²⁵I-labeled MAbs, 6×10⁴ cpm/dish. Afterward, thecultures were rinsed four times with PBS and solubilized with 1 ml/dishof 2 N NaOH, and the radioactivity was determined on a gamma counter.

[0270] The simple radioimmunoassay procedure of this example wasperformed directly in petri dish cultures. Sixteen variants of theradioimmunoassay enabled us to determine whether the MX and MN antigensare located on the surface or in the interior of the cells and how theexpression of those two antigens depends on infection with MaTu and onthe density, in which the cells had been grown before the petri disheswere seeded. In live, unfixed cells only cell surface antigens can bindthe MAbs. In those cells, M67 showed no reaction with any variant of thecultures, whereas M75 reacted in accord with the results of Examples 1and 2 above.

[0271] Fixation of the cells with methanol made the cell membranepermeable to the MAbs: M67 reacted with HeLa infected with MaTu,independently of previous cell density, and it did not bind to controlHeLa. MAb M75 in methanol-fixed cells confirmed the absence ofcorresponding antigen in uninfected HeLa from sparse cultures and itsinduction both by growth in dense cultures and by infection with MaTu.

EXAMPLE 4 Identification of MaTu Components Reactive with Animal Sera orAssociated with VSV Virions

[0272] Immunoblot analyses of MaTu-specific proteins from RIPA extractsfrom uninfected or MaTu-infected HeLa and from purified VSV reproducedin control or in MaTu-infected HeLa, identified which of the antigens,p58X or p54/58N, were radioimmunoprecipitated with animal sera, andwhich of them was responsible for complementation of VSV mutants and forthe formation of pseudotype virions. Details concerning the procedurescan be found in Pastorekova et al., Virology, 187: 620-626 (1992).

[0273] The serum of a rabbit immunized with MaTu-infected HeLaimmunoprecipitated both MAb M67- and MAb M75-reactive proteins (bothp58X and p54/58N), whereas the “spontaneously” immune sera of normalrabbit, sheep or leukemic cow immunoprecipitated only the M67-reactiveprotein (p58X). On the other hand, in VSV reproduced in MaTu-infectedHeLa cells and subsequently purified, only the M75-reactive bands ofp54/58N were present. Thus, it was concluded that MX and MN areindependent components of MaTu, and that it was p54/58N thatcomplemented VSV mutants and was assembled into pseudotype virions.

[0274] As shown in FIG. 6 discussed below in Example 5, MX antigen wasfound to be present in MaTu-infected fibroblasts. In Zavada andZavadova, supra, it was reported that a p58 band from MX-infectedfibroblasts could not be detected by RIP with rabbit anti-MaTu serum.That serum contains more antibodies to MX than to MN antigen. Thediscrepancy can be explained by the extremely slow spread of MX ininfected cultures. The results reported in Zavada and Zavadova, suprawere from fibroblasts tested 6 weeks after infection, whereas the latertesting was 4 months after infection. We have found by immunoblots thatMX can be first detected in both H/F-N and H/F-T hybrids after 4 weeks,in HeLa cells after six weeks and in fibroblasts only 10 weeks afterinfection.

EXAMPLE 5 Expression of MN- and MX-Specific Proteins

[0275]FIG. 6 graphically illustrates the expression of MN- andMX-specific proteins in human fibroblasts, in HeLa cells and in H/F-Nand H/F-T hybrid cells, and contrasts the expression in MX-infected anduninfected cells. Cells were infected with MX by co-cultivation withmitomycin C-treated MX-infected HeLa. The infected and uninfected cellswere grown for three passages in dense cultures. About four months afterinfection, the infected cells concurrently with uninfected cells weregrown in petri dishes to produce dense monolayers.

[0276] A radimmunoassay was performed directly in confluent petri dish(5 cm) culture of cells, fixed with methanol essentially as described inExample 3, supra. The monolayers were fixed with methanol and treatedwith 125I-labeled MAbs M67 (specific for exogenous MX antigen) or M75(specific for endogenous MN antigen) at 6×10⁴ cpm/dish. The boundradioactivity was measured; the results are shown in FIG. 6.

[0277]FIG. 6 shows that MX was transmitted to all four cell linestested, that is, to human embryo fibroblasts, to HeLa and to both H/F-Nand H/F-T hybrids; at the same time, all four uninfected counterpartcell lines were MX-negative (top graph of FIG. 6). MN antigens are shownto be present in both MX-infected and uninfected HeLa and H/F-T cells,but not in the fibroblasts (bottom graph of FIG. 6). No MN antigen wasfound in the control H/F-N, and only a minimum increase over backgroundof MN antigen was found in MaTu infected H/F-N. Thus, it was found thatin the hybrids, expression of MN antigen very strongly correlates withtumorigenicity.

[0278] Those results were consistent with the results obtained byimmunoblotting as shown in FIG. 7. The MN-specific twin protein p54/58Nwas detected in HeLa cell lines (both our standard type, that is, HeLaK, and in the Stanbridge mutant HeLa, that is, D98/AH.2 shown as HeLa S)and in tumorigenic H/F-T; however, p54/58N was not detected in thefibroblasts nor in the non-tumorigenic H/F-N even upon deliberately longexposure of the film used to detect radioactivity. Infection of the HeLacells with MX resulted in a strong increase in the concentration of thep54/58N protein(s).

[0279] The hybrid cells H/F-N and H/F-T were constructed by Eric J.Stanbridge [Stanbridge et al., Somatic Cell Genetics. 7: 699-712 (1981);and Stanbridge et al., Science. 215: 252-259 (1982)]. His originalhybrid, produced by the fusion of a HeLa cell and a human fibroblast wasnot tumorigenic in nude mice, although it retained some properties oftransformed cells, for example, its growth on soft agar. Rare segregantsfrom the hybrid which have lost chromosome 11 are tumorigenic. The mostlikely explanation for the tumorigenicity of those segregants is thatchromosome 11 contains a suppressor gene (an antioncogene), which blocksthe expression of a as yet unknown oncogene. The oncoprotein encoded bythat oncogene is critical for the capacity of the H/F hybrids to producetumors in nude mice. Since the p54/58N protein shows a correlation withthe tumorigenicity of H/F hybrids, it is a candidate for that putativeoncoprotein.

EXAMPLE 6 Immunoblots of MN Antigen from Human Tumor Cell Cultures andfrom Clinical Specimens of Human Tissues

[0280] The association of MN antigen with tumorigenicity in the H/Fhybrid cells as illustrated by Example 5 prompted testing for thepresence of MN antigen in other human tumor cell cultures and inclinical specimens. Preliminary experiments indicated that theconcentration of MN antigen in the extracts from other human tumor cellcultures was lower than in HeLa; thus, it was realized that longexposure of the autoradiographs would be required. Therefore, thesensitivity of the method was increased by the method indicated underMaterials and Methods: Immunoblotting, supra, wherein the MN antigen wasconcentrated by precipitation with MAb M75-loaded SAC.

[0281]FIG. 8 shows the immunoblots wherein lane A, a cell cultureextract from MX-infected HeLa cells was analysed directly (10 μl perlane) whereas the antigens from the other extracts (lanes B-E) were eachconcentrated from a 500 μl extract by precipitation with MAb M75 andSAC.

[0282]FIG. 8 indicates that two other human carcinoma cell lines containMN-related proteins—T24 (bladder carcinoma; lane C) and T47D (mammarycarcinoma; lane D). Those cells contain proteins which react with MAbM75 that under reducing conditions have molecular weights of 54 kd and56 kd, and under non-reducing conditions have a molecular weight ofabout 153 kd. The intensity of those bands is at least ten times lowerthan that for the p54/58N twin protein from HeLa cells.

[0283] An extremely weak band at approximately 52 kd could be seen underreducing conditions from extracts from human melanoma cells (SK-Mel1477; lane E), but no bands for human fibroblast extracts (lane B) couldbe seen either on the reducing or non-reducing gels.

[0284]FIG. 9 shows immunoblots of human tissue extracts includingsurgical specimens as compared to a cell extract from MX-infected HeLa(lane A). The MN-related antigen from all the extracts but for lane A(analysed directly at 10 μl per lane) was first concentrated from a 1 mlextract as explained above. MN proteins were found in endometrial (lanesD and M), ovarian (lanes E and N) and in uterine cervical (lane 0)carcinomas. In those extracts MN-related proteins were found in threebands having molecular weights between about 48 kd and about 58 kd.Another MN-related protein was present in the tissue extract from amammary papilloma that protein was seen as a single band at about 48 kd(lane J).

[0285] Clearly negative were the extracts from full-term placenta (laneB), normal mammary gland (lane K), hyperplastic endometrium (lane L),normal ovaries (lane H), and from uterine myoma (lane I). Only extremelyslightly MN-related bands were seen in extracts from trophoblasts (lanesF and G) and from melanoma (lane P).

[0286] The observations that antigen related to p54/58N was expressed inclinical specimens of several types of human carcinomas but not ingeneral in normal tissues of the corresponding organs (exceptionsdelineated in Example 13) further strengthened the association of MNantigen with tumorigenesis. However, it should be noted that for humantumors, a normal tissue is never really an adequate control in thattumors are believed not to arise from mature, differentiated cells, butrather from some stem cells, capable of division and of differentiation.In body organs, such cells may be quite rare.

EXAMPLE 7 MN Antigen in Animal Cell Lines

[0287] Since the MN gene is present in the chromosomal DNA of allvertebrate species that were tested, MN-related antigen was searched foralso in cell lines derived from normal tissues and from tumors ofseveral animal species. MN-related protein was found in two rat celllines: one of them was the XC cell line derived from ratrhabdomyosarcoma induced with Rous sarcoma virus; the other was theRat2-Tk⁻ cell line. In extracts from both of those rat cell lines, asingle protein band was found on the blots: its molecular weight onblots produced from a reducing gel and from a non-reducing gel wasrespectively 53.5 kd and 153 kd. FIG. 10 shows the results with Rat2-Tk⁻cell extracts (lane B), compared with extracts from MX-infected HeLa(lane A); the concentration of MN antigen in those two cell lines isvery similar. The extracts were analysed directly (40 μl per lane).

[0288] MN-related protein from XC cells showed the same pattern as forRat2-Tk⁻ cells both under reducing and non-reducing conditions, exceptthat its concentration was about 30× lower. The finding of a MN-relatedprotein—p53.5N—in two rat cell lines (FIGS. 10 and 12) provides thebasis for a model system.

[0289] None of the other animal cell lines tested contained detectableamounts of MN antigen, even when the highly sensitive immunoblottechnique in which the MN antigens are concentrated was used. TheMN-negative cells were: Vero cells (African green monkey); mouse Lcells; mouse NIH-3T3 cells normal, infected with Moloney leukemia virus,or transformed with Harvey sarcoma virus; GR cells (mouse mammary tumorcells induced with MTV), and NMG cells (normal mouse mammary gland).

EXAMPLE 8 Radioimmunoassays in Liquid Phase Using Recombinant MN Proteinfor MN-Specific Antibodies and for MN Antigen

[0290] The genetically engineered MN protein fused with glutathioneS-transferase—GEX-3X-MN—prepared and purified as described above waslabeled with ¹²⁵I by the chloramine T method [Hunter (1978)]. Thepurified protein enabled the development of a quantitative RIA forMN-specific antibodies as well as for MN antigens. All dilutions ofantibodies and of antigens were prepared in RIPA buffer (1% TRITON X-100and 0.1% sodium deoxycholate in PBS—phosphate buffered saline, pH 7.2),to which was added 1% of fetal calf serum (FCS). Tissue and cellextracts were prepared in RIPA buffer containing 1 mMphenylmethylsulfonylfluoride and 200 trypsin inhibiting units ofTrasylol (aprotinin) per ml, with no FCS. ¹²⁵I-labeled GEX-3X-MN protein(2.27 μCi/μg of TCA-precipitable protein) was before use diluted withRIPA+1% FCS, and non-specifically binding radioactivity was adsorbedwith a suspension of fixed protein A-Staphylococcus aureus cells (SAC).

[0291] In an RIA for MN-specific antibodies, MAb-containing ascitesfluids or test sera were mixed with ¹²⁵I-labeled protein and allowed toreact in a total volume of 1 ml for 2 hours at room temperature.Subsequently, 50 μl of a 10% suspension of SAC [Kessler, supra) wasadded and the mixture was incubated for 30 minutes. Finally, the SAC waspelleted, 3× washed with RIPA, and the bound radioactivity wasdetermined on a gamma counter.

[0292] Titration of antibodies to MN antigen is shown in FIG. 11.Ascitic fluid from a mouse carrying M75 hybridoma cells (A) is shown tohave a 50% end-point at dilution 1:1.4×10⁻⁶. At the same time, asciticfluids with MAbs specific for MX protein (M16 and M67) showed noprecipitation of. ¹²⁵I-labeled GEX-3X-MN even at dilution 1:200 (resultnot shown). Normal rabbit serum (C) did not significantly precipitatethe MN antigen; rabbit anti-MaTu serum (B), obtained after immunizationwith live MX-infected HeLa cells, precipitated 7% of radioactive MNprotein, when diluted 1:200. The rabbit anti-MaTu serum is shown byimmunoblot in Example 4 (above) to precipitate both MX and MN proteins.

[0293] Only one out of 180 human sera tested (90 control and 90 sera ofpatients with breast, ovarian or uterine cervical cancer) showed asignificant precipitation of the radioactively labeled MN recombinantprotein. That serum—L8—(D) was retested on immunoblot (as in Example 4),but it did not precipitate any p54/58N from MX-infected HeLa cells.Also, six other human sera, including KH (E), were negative onimmunoblot. Thus, the only positive human serum in the RIA, L8, wasreactive only with the genetically engineered product, but not withnative p54/58N expressed by HeLa cells.

[0294] In an RIA for MN antigen, the dilution of MAb M75, which in theprevious test precipitated 50% of maximum precipitable radioactivity(=dilution 1:1.4×10⁻⁶) was mixed with dilutions of cell extracts andallowed to react for 2 hours. Then, ¹²⁵I-labeled GEX-3X-MN (25×10³cpm/tube) was added for another 2 hours. Finally, the radioactivitybound to MAb M75 was precipitated with SAC and washed as above. Onehundred percent precipitation (=0 inhibition) was considered the maximumradioactivity bound by the dilution of MAb used. The concentration ofthe MN antigen in the tested cell extracts was calculated from aninhibition curve obtained with “cold” GEX-3X-MN, used as the standard (Ain FIG. 12).

[0295] The reaction of radioactively labeled GEX-3X-MN protein with MAbM75 enabled us to quantitate MN antigen directly in cell extracts. FIG.12 shows that 3 ng of “cold” GEX-3X-MN (A) caused a 50% inhibition ofprecipitation of “hot” GEX-3X-MN; an equivalent amount of MN antigen ispresent in 3×10³ ng of proteins extracted from MaTu-infected HeLa (B) orfrom Rat2-Tk⁻ cells (C). Concentrations of MN protein in cell extracts,determined by this RIA, are presented in Table 1 below. It must beunderstood that the calculated values are not absolute, since MNantigens in cell extracts are of somewhat different sizes, and alsosince the genetically engineered MN protein is a product containingmolecules of varying size. TABLE 1 Concentration of MN Protein in CellExtracts Cells ng MN/mg total protein HeLa + MX 939.00 Rat2-Tk⁻ 1065.00HeLa 27.50 XC 16.40 T24 1.18 HEF 0.00

[0296] The data were calculated from the results shown in FIG. 12.

EXAMPLE 9 Immunoelectron and Scanning Microscopy of Control and ofMX-infected HeLa Cells

[0297] As indicated above in Example 1, MN antigen, detected by indirectimmunofluorescence with MAb M75, is located on the surface membranes andin the nuclei of MX-infected HeLa cells or in HeLa cells grown in densecultures. To elucidate more clearly the location of the MN antigen,immunoelectron microscopy was used wherein MAb M75 bound to MN antigenwas visualized with immunogold beads. [Herzog et al., “Colloidal goldlabeling for determining cell surface area,” IN: Colloidal Gold, Vol. 3(Hayat, M A., ed.), pp. 139-149 (Academic Press Inc.; San Diego,Calif.).]

[0298] Ultrathin sections of control and of MX-infected HeLa cells areshown in FIG. 13A-D. Those immuno-electron micrographs demonstrate thelocation of MN antigen in the cells, and in addition, the strikingultrastructural differences between control and MX-infected HeLa. Acontrol HeLa cell (FIG. 13A) is shown to have on its surface very littleMN antigen, as visualised with gold beads. The cell surface is rathersmooth, with only two little protrusions. No mitochondria can be seen inthe cytoplasm. In contrast, MX-infected HeLa cells (FIGS. 13B and C)show the formation of abundant, dense filamentous protrusions from theirsurfaces. Most of the MN antigen is located on those filaments, whichare decorated with immunogold. The cytoplasm of MX-infected HeLacontains numerous mitochondria (FIG. 13C). FIG. 13D demonstrates thelocation of MN antigen in the nucleus: some of the MN antigen is innucleoplasm (possibly linked to chromatin), but a higher concentrationof the MN antigen is in the nucleoli. Again, the surface of normal HeLa(panels A and E of FIG. 13) is rather smooth whereas MX-infected HeLacells have on their surface, numerous filaments and “blebs”. Some of thefilaments appear to form bridges connecting them to adjacent cells.

[0299] It has been noted that in some instances of in vitro transformedcells compared to their normal parent cells that one of the differencesis that the surface of normal cells was smooth whereas on thetransformed cells were numerous hair-like protrusions [Darnell et al.“Molecular Cell Biology,” (2nd edition) Sci. Am. Books; W.H. Freeman andCo., New York (1990)]. Under that criteria MX-infected HeLa cells, asseen in FIG. 13F, has a supertransformed appearance.

[0300] Further in some tumors, amplification of mitochondria has beendescribed [Bernhard, W., “Handbook of Molecular Cytology,” pp. 687-715,Lima de Faria (ed.), North Holland Publishing Co.; Amsterdam-London(1972)]. Such amplification was noted for MX-infected HeLa cells whichstained very intensely with Janus' green, specific for mitochondriawhereas control HeLa were only weakly stained.

[0301] It should be noted that electron microscopists were unable tofind any structural characteristics specific for tumor cells.

EXAMPLE 10 Antisense ODNs Inhibit MN Gene Expression

[0302] To determine whether both of the p54/58N proteins were encoded byone gene, the following experiments with antisense ODNs were performed.Previously sparse-growing HeLa cells were seeded to obtain anovercrowded culture and incubated for 130 hours either in the absence orin the presence of two gene-specific ODNs complementary to the 5′ end ofMN mRNA. HeLa cells were subcultured at 8×10⁵ cells per ml of DMEM with10% FCS. Simultaneously, ODNs were added to the media as follows: (A)29-mer ODN1 (5′ CGCCCAGTGGGTCATCTTCCCCAGAAGAG 3′ [SEQ. ID. NO.: 3),complementary to positions 44-72 of FIG. 1A-1B) in 4 μM finalconcentration, (B) 19-mer ODN2 (5′ GGAATCCTCCTGCATCCGG 3′ [SEQ. ID. NO.:4], complementary to positions 12-30) in 4 μM final concentration and(C) both ODN1 and ODN2 in 2 μM final concentration each. (D) Cellstreated in the same way, but incubated without ODNs, served as acontrol. After 130 hours, extracts from the cells were prepared andanalyzed by immunoblotting using ¹²⁵I-labeled MAb M75. Protein extractsfrom the cells were analyzed by immunoblotting and RIA using MAb M75.FIG. 3 provides the immunoblot results of those experiments.

[0303] It was found that cultivation of HeLa cells with the ODNsresulted in considerable inhibition of p54/58N synthesis. The 19-merODN2 (FIG. 3B) in 4 μM final concentration was very effective; asdetermined by RIA, it caused 40% inhibition, whereas the 29-mer ODN1 (4μM) (FIG. 3A) and a combination of the two ODNs (FIG. 3C), each in 2 μMfinal concentration, were less effective in RIA showing a 25-35%increase of the MN-related proteins. At the same time, the amount ofdifferent HeLa cell protein determined by RIA using specific MAb H460was in all cell variants approximately the same. Most importantly wasthat on immunoblot it could be seen that specific inhibition by the ODNsaffected both of the p54/58N proteins. Thus, we concluded that the MNgene we cloned coded for both p54/58N proteins in HeLa cells.

[0304] The results indicated that the MN twin proteins arise bytranslation of a single mRNA (consistent with the Northern blottingdata). Thus, the twin proteins may represent either differences inpost-translational modification (phosphorylation, protease processing,etc.), or the use of alternative translational initiation sites.

EXAMPLE 11 Northern Blotting of MN mRNA in Tumorigenic andNon-Tumorigenic Cell Lines

[0305]FIG. 4 shows the results of Northern blotting of MN mRNA in humancell lines. Total RNA was prepared from the following cell lines by theguanidinium thiocyanate-CsCl method: HeLa cells growing in a dense (A)and sparse (B) culture; CGL1 (H/F-N) hybrid cells (C); CGL3 (D) and CGL4(E) segregants (both H/F-T); and human embryo fibroblasts (F). Fifteenμg of RNA were separated on a 1.2% formaldehyde gel and blotted onto aHybond C Super membrane [Amersham]. MN cDNA NotI probe was labeled byrandom priming [Multiprime DNA labelling system; Amersham].Hybridization was carried out in the presence of 50% formamide at 42°C., and the final wash was in 0.1% SSPE and 0.1% SDS at 65° C. An RNAladder (0.24-9.5 kb) [BRL; Bethesda, Md. (USA)]was used as a sizestandard. Membranes were exposed to films at −70° C., with intensifyingscreens.

[0306] Detected was a 1.5 kb MN-specific mRNA only in two tumorigenicsegregant clones—CGL3 and CGL4 (H/F-T), but not in the non-tumorigenichybrid clone CGL1 (H/F-N) or in normal human fibroblasts. Further, the1.5 kb mRNA was found in the HeLa cells growing in dense (FIG. 4A) butnot in sparse (FIG. 4B) culture.

[0307] Thus, the results of the Northern blotting were consistent withother examples in regard to MN-related proteins being associated withtumorigenicity.

EXAMPLE 12 Southern Blotting of Genomic DNAs from Different VertebrateSpecies to Detect MN Gene and Restriction Analysis of Genomic DNA ofHeLa Cells

[0308]FIG. 5 illustrates the detection of MN genes in the genomic DNAsof various vertebrates by Southern blotting. Chromosomal DNA digested byPstI was as follows: (A) chicken; (B) bat; (C) rat; (D) mouse; (E)feline; (F) pig; (G) sheep; (H) bovine; (I) monkey; and (J) human HeLacells. Restriction fragments were separated on a 0.7% agarose gel andalkali blotted onto a Hybond N membrane (Amersham]. The MN cDNA probelabelling and hybridization procedures were the same as for the Northernblotting analyses shown in FIG. 4 and described in Example 11. TheSouthern blot of FIG. 5 made with PstI indicates that the MN gene isconserved in a single copy in all vertebrate genomes tested.

[0309] HeLa. Further, genomic DNA from HeLa cells was prepared asdescribed by Ausubel et al., Short Protocols in Molecular Biology[Greene Publishing Associates and Wiley-Interscience; New York (1989)],digested with different restriction enzymes, resolved on an agarose geland transferred to Hybond N+ membrane [Amersham]. The HeLa genomic DNAwas cleaved with the following restriction enzymes with the resultsshown in FIG. 17 (wherein the numbers in parentheses after the enzymesindicate the respective lanes in FIG. 17): EcoRI (1), EcoRV (2), HindIII(3), KpnI (4), NcoI (5), PstI (6), and PvuII (7), and then analyzed bySouthern hybridization under stringent conditions using MN cDNA as aprobe.

[0310] The prehybridization and hybridization using an MN cDNA probelabelled with ³²p-dCTP by random priming [Multi-prime DNA labellingsystem; Amersham] as well as wash steps were carried out according toAmersham's protocols at high stringency. A 1 kb DNA Ladder [from BRL;Bethesda, Md. (USA)] was used as a size standard. Membranes were exposedto films at −70° C., with intensifying screens.

[0311] The Southern blotting analysis of HeLa chromosomal DNA showedthat the gene coding for MN is present in the human genome in a singlecopy (FIG. 17). The sizes and distribution of MN-positive restrictionfragments obtained using the restriction enzymes KpnI, NcoI and HindIIIindicate that the MN gene contains introns, since those enzymes cut theMN genomic sequences despite the absence of their restriction sites inMN cDNA.

EXAMPLE 13 Immunohistochemical Staining of Tissue Specimens

[0312] To study and evaluate the tissue distribution range andexpression of MN proteins, the monoclonal antibody M75 was used to stainimmunohistochemically a variety of human tissue specimens. The primaryantibody used in these immunohistochemical staining experiments was theM75 monoclonal antibody. A biotinylated second antibody andstreptavidin-peroxidase were used to detect the M75 reactivity insections of formalin-fixed, paraffin-embedded tissue samples. Acommercially available amplification kit, specifically the DAKO LSAB™kit [DAKO Corp., Carpinteria, Calif. (USA)] which provides matched,ready made blocking reagent, secondary antibody andsteptavidin-horseradish peroxidase was used in these experiments.

[0313] M75 immunoreactivity was tested according to the methods of thisinvention in multiple-tissue sections of breast, colon, cervical, lungand normal tissues. Such multiple-tissue sections were cut from paraffinblocks of tissues called “sausages” that were purchased from the City ofHope [Duarte, Calif. (USA)]. Combined in such a multiple-tissue sectionwere normal, benign and malignant specimens of a given tissue; forexample, about a score of tissue samples of breast cancers fromdifferent patients, a similar number of benign breast tissue samples,and normal breast tissue samples would be combined in one suchmultiple-breast-tissue section. The normal multiple-tissue sectionscontained only normal tissues from various organs, for example, liver,spleen, lung, kidney, adrenal gland, brain, prostate, pancreas, thyroid,ovary, and testis.

[0314] Also screened for MN gene expression were multiple individualspecimens from cervical cancers, bladder cancers, renal cell cancers,and head and neck cancers. Such specimens were obtained from U.C. DavisMedical Center in Sacramento, Calif. and from Dr. Shu Y. Liao[Department of Pathology; St. Joseph Hospital; Orange, Calif. (USA)].

[0315] Controls used in these experiments were the cell lines CGL3(H/F-T hybrid cells) and CGL1 (H/F-N hybrid cells) which are known tostain respectively, positively and negatively with the M75 monoclonalantibody. The M75 monoclonal antibody was diluted to a 1:5000 dilutionwherein the diluent was either PBS [0.05 M phosphate buffered saline(0.15 M NaCl), pH 7.2-7.4] or PBS containing 1% protease-free BSA as aprotein stabilizer.

[0316] Immunohistochemical Staining Protocol

[0317] The immunohistochemical staining protocol was followed accordingto the manufacturer's instructions for the DAKO LSAB™ kit. In brief, thesections were dewaxed, rehydrated and blocked to remove non-specificreactivity as well as endogenous peroxidase activity. Each section wasthen incubated with dilutions of the M75 monoclonal antibody. After theunbound M75 was removed by rinsing the section, the section wassequentially reacted with a biotinylated antimouse IgG antibody andstreptavidin conjugated to horseradish peroxidase; a rinsing step wasincluded between those two reactions and after the second reaction.Following the last rinse, the antibody-enzyme complexes were detected byreaction with an insoluble chromogen (diaminobenzidine) and hydrogenperoxide. A positive result was indicated by the formation of aninsoluble reddish-brown precipitate at the site of the primary antibodyreaction. The sections were then rinsed, counterstained withhematoxylin, dehydrated and cover slipped. Then the sections wereexamined using standard light microscopy. The following is an outline ofexemplary steps of the immunohistochemical staining protocol.  1. Seriesof ETOH-baths 100, 100, 95, 2 min. ± 1 min.    95, 70% each  2. dH₂0wash - 2x 2 min. ± 1 min. each  3. 3% H₂0₂ as endogenous peroxidaseblock 5 min.  4. PBS wash - 2x 2 min. ± 1 min.  5. normal serum block(1.5% NGS) 30 min.  6. primary antibody (Mab M75) 60 min. ± 5 min.  7.PBS wash - 2x 2 min. ± 1 min.  8. biotinylated secondary antibody 20-30min. ± 2 min.  9. PBS wash - 2x 2 min. ± 1 min. 10.streptavidin-peroxidase reagent 20-30 min. ± 2 min. 11. PBS wash - 2x 2min. ± 1 min. 12. DAB (150 ml Tris, 90 μl H₂0₂, 3 ml KPL 5-6 min.   DAB) 13. PBS rinse, dH₂0 wash 1-2 min. 14. Hematoxylin counterstain 2min. ± 1 min. 15. wash with running tap water until clear 16. 0.05%ammonium hydroxide 20 sec. ± 10 sec. 17. dH₂0 wash - 2x 3 min. ± 1 min.18. dehydrate 70, 95, 95, 100, 100% EtOH 2 min. ± 1 min. each 19. xylene3x 3 min. ± 1 min. each 20. coverslip with Permount ™ [Fisher   Scientific Pittsburgh, PA (USA)] 21. wait 10 min. before viewingresults.

[0318] Interpretation. A deposit of a reddish brown precipitate over theplasma membrane was taken as evidence that the M75 antibody had bound toa MN antigen in the tissue. The known positive control (CGL3) had to bestained to validate the assay. Section thickness was taken intoconsideration to compare staining intensities, as thicker sectionsproduce greater staining intensity independently of other assayparameters.

[0319] The above-described protocol was optimized for formalin-fixedtissues, but can be used to stain tissues prepared with other fixatives.

[0320] Results

[0321] Preliminary examination of cervical specimens showed that 62 of68 squamous cell carcinoma specimens (91.2%) stained positively withM75. Additionally, 2 of 6 adenocarcinomas and 2 of 2 adenosquamouscancers of the cervix also stained positively. In early studies, 55.6%(10 of 18) of cervical dysplasias stained positively. A total of 9specimens including both cervical dysplasias and tumors, exhibited someMN expression in normal appearing areas of the endocervical glandularepithelium, usually at the basal layer. In some specimens, whereasmorphologically normal-looking areas showed expression of MN antigen,areas exhibiting dysplasia and/or malignancy did not show MN expression.

[0322] M75 positive immunoreactivity was most often localized to theplasma membrane of cells, with the most apparent stain being present atthe junctions between adjacent cells. Cytoplasmic staining was alsoevident in some cells; however, plasma membrane staining was most oftenused as the main criterion of positivity.

[0323] M75 positive cells tended to be near areas showing keratindifferentiation in cervical specimens. In some specimens, positivestaining cells were located in the center of nests of non-stainingcells. Often, there was very little, if any, obvious morphologicaldifference between staining cells and non-staining cells. In somespecimens, the positive staining cells were associated with adjacentareas of necrosis.

[0324] In most of the squamous cell carcinomas of the cervix, the M75immunoreactivity was focal in distribution, i.e., only certain areas ofthe specimen stained. Although the distribution of positive reactivitywithin a given specimen was rather sporadic, the intensity of thereactivity was usually very strong. In most of the adenocarcinomas ofthe cervix, the staining pattern was more homogeneous, with the majorityof the specimen staining positively.

[0325] Among the normal tissue samples, intense, positive and specificM75 immunoreactivity was observed only in normal stomach tissues, withdiminishing reactivity in the small intestine, appendix and colon. Noother normal tissue stained extensively positively for M75.Occasionally, however, foci of intensely staining cells were observed innormal intestine samples (usually at the base of the crypts) or weresometimes seen in morphologically normal appearing areas of theepithelium of cervical specimens exhibiting dysplasia and/or malignancy.In such, normal appearing areas of cervical specimens, positive stainingwas seen in focal areas of the basal layer of the ectocervicalepithelium or in the basal layer of endocervical glandular epithelium.In one normal specimen of human skin, cytoplasmic MN staining wasobserved in the basal layer. The basal layers of these epithelia areusually areas of proliferation, suggesting the MN expression may beinvolved in cellular growth. In a few cervical biopsied specimens, MNpositivity was observed in the morphologically normal appearingstratified squamous epithelium, sometimes associated with cellsundergoing koilocytic changes.

[0326] Some colon adenomas (4 of 11) and adenocarcinomas (9 of 15) werepositively stained. One normal colon specimen was positive at the baseof the crypts. Of 15 colon cancer specimens, 4 adenocarcinomas and 5metastatic lesions were MN positive. Fewer malignant breast cancers (3of 25) and ovarian cancer specimens (3 of 15) were positively stained.Of 4 head and neck cancers, 3 stained very intensely with M75.

[0327] Although normal stomach tissue was routinely positive, 4adenocarcinomas of the stomach were MN negative. Of 3 bladder cancerspecimens (1 adenocarcinoma, 1 non-papillary transitional cellcarcinoma, and 1 squamous cell carcinoma), only the squamous cellcarcinoma was MN positive. Approximately 40% (12 of 30) of lung cancerspecimens were positive; 2 of 4 undifferentiated carcinomas; 3 of 8adenocarcinomas; 2 of 8 oat cell carcinomas; and, 5 of 10 squamous cellcarcinomas. One hundred percent (4 of 4) of the renal cell carcinomaswere MN positive.

[0328] In summary, MN antigen, as detected by M75 andimmunohistochemistry in the experiments described above, was shown to beprevalent in tumor cells, most notably in tissues of cervical cancers.MN antigen was also found in some cells of normal tissues, and sometimesin morphologically normal appearing areas of specimens exhibitingdysplasia and/or malignancy. However, MN is not usually extensivelyexpressed in most normal tissues, except for stomach tissues where it isextensively expressed and in the tissues of the lower gastrointestinaltract where it is less extensively expressed. MN expression is mostoften localized to the cellular plasma membrane of tumor cells and mayplay a role in intercellular communication or cell adhesion.Representative results of experiments performed as described above aretabulated in Table 2. TABLE 2 Immunoreactivity of M75 in Various TissuesPOS/NEG TISSUE TYPE (#pos/#tested) liver, spleen, lung, normal NEG (all)kidney, adrenal gland, brain, prostate, pancreas, thyroid ovary, testisskin normal POS (in basal layer) (1/1) stomach normal POS smallintestine normal POS colon normal POS breast normal NEG (0/10) cervixnormal NEG (0/2) breast benign NEG (0/17) colon benign POS (4/11) cervixbenign POS (10/18) breast malignant POS (3/25) colon malignant POS(9/15) ovarian malignant POS (3/15) lung malignant POS (12/30) bladdermalignant POS (1/3) head & neck malignant POS (3/4) kidney malignant POS(4/4) stomach malignant NEG (0/4) cervix malignant POS (62/68)

[0329] The results recorded in this example indicate that the presenceof MN proteins in a tissue sample from a patient may, in general,depending upon the tissue involved, be a marker signaling that apre-neoplastic or neoplastic process is occurring. Thus, one mayconclude from these results that diagnostic/prognostic methods thatdetect MN antigen may be particularly useful for screening patientsamples for a number of cancers which can thereby be detected at apre-neoplastic stage or at an early stage prior to obvious morphologicchanges associated with dysplasia and/or malignancy being evident orbeing evident on a widespread basis.

EXAMPLE 14 Vaccine—Rat Model

[0330] As shown above in Example 7, in some rat tumors, for example, theXC tumor cell line (cells from a rat rhabdomyosarcoma), a rat MNprotein, related to human MN, is expressed. Thus a model was afforded tostudy antitumor immunity induced by experimental MN-based vaccines. Thefollowing representative experiments were performed.

[0331] Nine- to eleven-day-old Wistar rats from several families wererandomized, injected intraperitoneally with 0.1 ml of either control ratsera (the C group) or with rat serum against the MN fusion proteinGEX-3X-MN (the IM group). Simultaneously both groups were injectedsubcutaneously with 10⁶ XC tumor cells.

[0332] Four weeks later, the rats were sacrificed, and their tumorsweighed. The results are shown in FIG. 14. Each point on the graphrepresents a tumor from one rat. The difference between the two groups—Cand IM—was significant by Mann-Whitney rank test (U=84, α (0.025). Theresults indicate that the IM group of baby rats developed tumors aboutone-half the size of the controls, and 5 of the 18 passively immunizedrats developed no tumor at all, compared to 1 of 18 controls.

EXAMPLE 15 Expression of Full-Length MN cDNA in NIH 3T3 Cells

[0333] The role of MN in the regulation of cell proliferation wasstudied by expressing the full-length cDNA in NIH 3T3 cells. That cellline was chosen since it had been used successfully to demonstrate thephenotypic effect of a number of proto-oncogenes [Weinberg, R. A.,Cancer Res., 49: 3713 (1989); Hunter, T., Cell, 64: 249 (1991)]. Also,NIH 3T3 cells express no endogenous MN-related protein that isdetectable by Mab M75.

[0334] The full length MN cDNA was obtained by ligation of the two cDNAclones using the unique BamHI site and subcloned from pBluescript intoKpnI-SacI sites of the expression vector pSG5C. pSG5C was kindlyprovided by Dr. Richard Kettman [Department of Molecular Biology,Faculty of Agricultural Sciences, B-5030 Gembloux, Belgium]. pSG5C wasderived from pSG5 [Stratagene] by inserting a polylinker consisting of asequence having several neighboring sites for the following restrictionenzymes: EcoRI, XhoI, KpnI, BamHI, SacI, 3 times TAG stop codon andBglII.

[0335] The recombinant pSG5C-MN plasmid was co-transfected in a 10:1ratio (10 μg: 1 μg) with the pSV2neo plasmid [Southern and Berg, J. Mol.Appl. Genet., 1: 327 (1982)] which contains the neo gene as a selectionmarker. The co-transfection was carried out by calcium phosphateprecipitation method [Mammalian Transfection Kit; Stratagene] into NIH3T3 cells plated a day before at a density of 1×10⁵ per 60 mm dish. As acontrol, pSV2neo was co-transfected with empty pSG5C.

[0336] Transfected cells were cultured in DMEM medium supplemented with10% FCS and 600 μg ml⁻¹ of G418 [Gibco BRL] for 14 days. TheG418-resistant cells were clonally selected, expanded and analysed forexpression of the transfected cDNA by Western blotting using iodinatedMab M75.

[0337] For an estimation of cell proliferation, the clonal cell lineswere plated in triplicates (2×10⁴ cells/well) in 24-well plates andcultivated in DMEM with 10% FCS and 1% FCS, respectively. The medium waschanged each day, and the cell number was counted using a hemacytometer.

[0338] To determine the DNA synthesis, the cells were plated intriplicate in 96-well plate at a density of 10⁴/well in DMEM with 10%FCS and allowed to attach overnight. Then the cells were labeled with³H-thymidine for 24 hours, and the incorporated radioactivity wascounted.

[0339] For the anchorage-independent growth assay, cells (2×10⁴) weresuspended in a 0.3% agar in DMEM containing 10% FCS and overlaid onto0.5% agar medium in 60 mm dish. Colonies grown in soft agar were countedtwo weeks after plating.

[0340] Several clonal cell lines constitutively expressing both 54 and58 kd forms of MN protein in levels comparable to those found inLCMV-infected HeLa cells were obtained. Selected MN-positive clones andnegative control cells (mock-transfected with an empty pSG5C plasmid)were subjected to further analyses directed to the characterization oftheir phenotype and growth behavior.

[0341] The MN-expressing NIH 3T3 cells displayed spindle-shapedmorphology, and increased refractility; they were less adherent to thesolid support and smaller in size. The control (mock transfected cells)had a flat morphology, similar to parental NIH 3T3 cells. In contrast tothe control cells that were aligned and formed a monolayer with anordered pattern, the cells expressing MN lost the capacity for growtharrest and grew chaotically on top of one another (FIG. 23a-d).Correspondingly, the MN-expressing cells were able to reachsignificantly higher (more than 2×) saturation densities (Table 3) andwere less dependent on growth factors than the control cells (FIG.23g-h).

[0342] MN transfectants also showed faster doubling times (by 15%) andenhanced DNA synthesis (by 10%), as determined by the amount of[³H]-thymidine incorporated in comparison to control cells. Finally, NIH3T3 cells expressing MN protein grew in soft agar. The diameter ofcolonies grown for 14 days ranged from 0.1 to 0.5 mm (FIG. 23f);however, the cloning efficiency of MN transfectants was rather low(2.4%). Although that parameter of NIH 3T3 cells seems to be lessaffected by MN than by conventional oncogenes, all other data areconsistent with the idea that MN plays a role in cell growth control.TABLE 3 Growth Properties of NIH 3T3 Cells Expressing MN ProteinTransfected pSG5C/ pSG5C-MN/ DNA pSV2neo pSV2neo Doubling time^(a) 27.9± 0.5 24.1 ± 1.3 (hours) Saturation density^(b)  4.9 ± 0.2 11.4 ± 0.4(cells × 10⁴/cm²) Cloning <0.01  2.4 ± 0.2 efficiency (%)^(c)

EXAMPLE 16 Acceleration of G1 Transit and Decrease in Mitomycin CSensitivity Caused by MN Protein

[0343] For the experiments described in this example, the stable MNtransfectants of NIH 3T3 cells generated as described in Example 15 wereused. Four selected MN-positive clones and four control mock-transfectedclones were either used individually or in pools.

[0344] Flow cytometric analyses of asynchronous cell populations. Forthe results shown in FIG. 24(a), cells that had been grown in denseculture were plated at 1×10⁶ cells per 60 mm dish. Four days later, thecells were collected by trypsinization, washed, resuspended in PBS,fixed by dropwise addition of 70% ethanol and stained by propidiumiodine solution containing RNase. Analysis was performed by FACStarusing DNA cell cycle analysis software. [Becton Dickinson; FranklinLakes, N.J. (USA)].

[0345] For the analyses shown in FIG. 24(b) and (c), exponentiallygrowing cells were plated at 5×10⁵ cells per 60 mm dish and analysed asabove 2 days later. Forward light scatter was used for the analysis ofrelative cell sizes. The data were evaluated using Kolmogorov-Smirnovtest [Young, J. Histochem. Cytochem. 25: 935 (1977)]. D is the maximumdifference between summation curves derived from histograms. D/s(n) is avalue which indicates the similarity of the compared curves (it is closeto zero when curves are similar).

[0346] The flow cytometric analyses revealed that clonal populationsconstitutively expressing MN protein showed a decreased percentage ofcells in G1 phase and an increased percentage of cells in G2-M phases.Those differences were more striking in cell populations grownthroughout three passages in high density cultures [FIG. 24(a)], than inexponentially growing subconfluent cells [FIG. 24(b)]. That observationsupports the idea that MN protein has the capacity to perturb contactinhibition.

[0347] Also observed was a decrease in the size of MN expressing cellsseen in both exponentially proliferating and high density cultures. Itis possible that the MN-mediated acceleration of G1 transit is relatedto the above-noted shorter doubling time (by about 15%) of exponentiallyproliferating MN-expressing NIH 3T3 cells. Also, MN expressing cellsdisplayed substantially higher saturation density and lower serumrequirements than the control cells. Those facts suggest thatMN-transfected cells had the capacity to continue to proliferate despitespace limitations and diminished levels of serum growth factors, whereasthe control cells were arrested in G1 phase.

[0348] Limiting conditions. The proliferation of MN-expressing andcontrol cells was studied both in optimal and limiting conditions. Cellswere plated at 2×10⁴ per well of 24-well plate in DMEM with 10% FCS. Themedium was changed at daily intervals until day 4 when confluency wasreached, and the medium was no longer renewed. Viable cells were countedin a hemacytometer at appropriate times using trypan blue dye exclusion.The numbers of cells were plotted versus time wherein each plot pointrepresents a mean value of triplicate determination.

[0349] The results showed that the proliferation of MN expressing andcontrol cells was similar during the first phase when the medium wasrenewed daily, but that a big difference in the number of viable cellsoccurred after the medium was not renewed. More than half of the controlcells were not able to withstand the unfavorable growth conditions. Incontrast, the MN-expressing cells continued to proliferate even whenexposed to increasing competition for nutrients and serum growthfactors.

[0350] Those results were supported also by flow cytometric analysis ofserum starved cells grown for two days in medium containing 1% FCS.While 83% of control cells accumulated in G0-G1 phase (S=5%, G2−M=12%),expression of MN protein partially reversed the delay in G1 as indicatedby cell cycle distribution of MN tranfectants (G0-G1=65%, S=10%,G2−M=26%). The results of the above-described experiments suggest thatMN protein might function to release the G1/S checkpoint and allow cellsto proliferate under unfavorable conditions.

[0351] MMC. To test that assumption, unfavorable conditions weresimulated by treating cells with the DNA damaging drug mitomycin C (MMC)and then following their proliferation and viability. The mechanism ofaction of MMC is thought to result from its intracellular activation andsubsequent DNA alkylation and crosslinking [Yier and Szybalski, Science,145: 55 (1964)]. Normally, cells respond to DNA damage by arrest oftheir cell cycle progression to repair defects and prevent acquisitionof genomic instability. Large damage is accompanied by markedcytotoxicity. However, many studies [for example, Peters et al., Int. J.Cancer, 54: 450 (1993)] concern the emergence of drug resistant cellsboth in tumor cell populations and after the introduction of oncogenesinto nontransformed cell lines.

[0352] The response of MN-transfected NIH 3T3 cells to increasingconcentrations of MMC was determined by continuous [³H]-thymidinelabeling. Cells were plated in 96-well microtiter plate concentration of10⁴ per well and incubated overnight in DMEM with 10% FCS to attach.Then the growth medium was replaced with 100 μl of medium containingincreasing concentrations of MMC from 1 μl/ml to 32 μg/ml. All the drugconcentrations were tested in three replicate wells. After 5 hours oftreatment, the MMC was removed, cells were washed with PBS and freshgrowth medium without the drug was added. After overnight recovery, thefractions of cells that were actively participating in proliferation wasdetermined by continuous 24-hr labeling with [³H]-thymidine. Theincorporation by the treated cells was compared to that of the control,untreated cells, and the proliferating fractions were considered as apercentage of the control's incorporation.

[0353] The viability of the treated cells was estimated three days laterby a CellTiter 96 AQ Non-Radioactive Cell Proliferation Assay (Promega]which is based on the bioreduction of methotrexate (MTX) into a watersoluble formazan that absorbs light at 490 nm. The percentage ofsurviving cells was derived from the values of absorbance obtained aftersubstraction of background.

[0354] The control and MN-expressing NIH 3T3 cells showed remarkabledifferences in their responses to MMC. The sensitivity of theMN-transfected cells appeared considerably lower than the control's inboth sections of the above-described experiments. The results suggestedthat the MN-transfected cells were able to override the negative growthsignal mediated by MMC.

[0355] ATCC Deposits. The material listed below was deposited with theAmerican Type Culture Collection (ATCC) at 12301 Parklawn Drive,Rockville, Md. 20852 (USA). The deposits were made under the provisionsof the Budapest Treaty on the International Recognition of DepositedMicroorganisms for the Purposes of Patent Procedure and Regulationsthereunder (Budapest Treaty). Maintenance of a viable culture is assuredfor thirty years from the date of deposit. The organism will be madeavailable by the ATCC under the terms of the Budapest Treaty, andsubject to an agreement between the Applicants and the ATCC whichassures unrestricted availability of the deposited hybridomas to thepublic upon the granting of patent from the instant application.Availability of the deposited strain is not to be construed as a licenseto practice the invention in contravention of the rights granted underthe authority of any Government in accordance with its patent laws.Hybridoma Deposit Date ATCC # VU-M75 Sep. 17, 1992 HB 11128 MN 12.2.2Jun. 9, 1994 HB 11647

[0356] The description of the foregoing embodiments of the inventionhave been presented for purposes of illustration and description. Theyare not intended to be exhaustive or to limit the invention to theprecise form disclosed, and obviously many modifications and variationsare possible in light of the above teachings. The embodiments werechosen and described in order to explain the principles of the inventionand its practical application to enable thereby others skilled in theart to utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

[0357] All references cited herein are hereby incorporated by reference.

1 26 1 1399 DNA HUMAN CDS (1)..(1266) 1 cag agg ttg ccc cgg atg cag gaggat tcc ccc ttg gga gga ggc tct 48 Gln Arg Leu Pro Arg Met Gln Glu AspSer Pro Leu Gly Gly Gly Ser 1 5 10 15 tct ggg gaa gat gac cca ctg ggcgag gag gat ctg ccc agt gaa gag 96 Ser Gly Glu Asp Asp Pro Leu Gly GluGlu Asp Leu Pro Ser Glu Glu 20 25 30 gat tca ccc aga gag gag gat cca cccgga gag gag gat cta cct gga 144 Asp Ser Pro Arg Glu Glu Asp Pro Pro GlyGlu Glu Asp Leu Pro Gly 35 40 45 gag gag gat cta cct gga gag gag gat ctacct gaa gtt aag cct aaa 192 Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu ProGlu Val Lys Pro Lys 50 55 60 tca gaa gaa gag ggc tcc ctg aag tta gag gatcta cct act gtt gag 240 Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp LeuPro Thr Val Glu 65 70 75 80 gct cct gga gat cct caa gaa ccc cag aat aatgcc cac agg gac aaa 288 Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn AlaHis Arg Asp Lys 85 90 95 gaa ggg gat gac cag agt cat tgg cgc tat gga ggcgac ccg ccc tgg 336 Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly Gly AspPro Pro Trp 100 105 110 ccc cgg gtg tcc cca gcc tgc gcg ggc cgc ttc cagtcc ccg gtg gat 384 Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe Gln SerPro Val Asp 115 120 125 atc cgc ccc cag ctc gcc gcc ttc tgc ccg gcc ctgcgc ccc ctg gaa 432 Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala Leu ArgPro Leu Glu 130 135 140 ctc ctg ggc ttc cag ctc ccg ccg ctc cca gaa ctgcgc ctg cgc aac 480 Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu Leu ArgLeu Arg Asn 145 150 155 160 aat ggc cac agt gtg caa ctg acc ctg cct cctggg cta gag atg gct 528 Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro GlyLeu Glu Met Ala 165 170 175 ctg ggt ccc ggg cgg gag tac cgg gct ctg cagctg cat ctg cac tgg 576 Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln LeuHis Leu His Trp 180 185 190 ggg gct gca ggt cgt ccg ggc tcg gag cac actgtg gaa ggc cac cgt 624 Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr ValGlu Gly His Arg 195 200 205 ttc cct gcc gag atc cac gtg gtt cac ctc agcacc gcc ttt gcc aga 672 Phe Pro Ala Glu Ile His Val Val His Leu Ser ThrAla Phe Ala Arg 210 215 220 gtt gac gag gcc ttg ggg cgc ccg gga ggc ctggcc gtg ttg gcc gcc 720 Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu AlaVal Leu Ala Ala 225 230 235 240 ttt ctg gag gag ggc ccg gaa gaa aac agtgcc tat gag cag ttg ctg 768 Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser AlaTyr Glu Gln Leu Leu 245 250 255 tct cgc ttg gaa gaa atc gct gag gaa ggctca gag act cag gtc cca 816 Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly SerGlu Thr Gln Val Pro 260 265 270 gga ctg gac ata tct gca ctc ctg ccc tctgac ttc agc cgc tac ttc 864 Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser AspPhe Ser Arg Tyr Phe 275 280 285 caa tat gag ggg tct ctg act aca ccg ccctgt gcc cag ggt gtc atc 912 Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro CysAla Gln Gly Val Ile 290 295 300 tgg act gtg ttt aac cag aca gtg atg ctgagt gct aag cag ctc cac 960 Trp Thr Val Phe Asn Gln Thr Val Met Leu SerAla Lys Gln Leu His 305 310 315 320 acc ctc tct gac acc ctg tgg gga cctggt gac tct cgg cta cag ctg 1008 Thr Leu Ser Asp Thr Leu Trp Gly Pro GlyAsp Ser Arg Leu Gln Leu 325 330 335 aac ttc cga gcg acg cag cct ttg aatggg cga gtg att gag gcc tcc 1056 Asn Phe Arg Ala Thr Gln Pro Leu Asn GlyArg Val Ile Glu Ala Ser 340 345 350 ttc cct gct gga gtg gac agc agt cctcgg gct gct gag cca gtc cag 1104 Phe Pro Ala Gly Val Asp Ser Ser Pro ArgAla Ala Glu Pro Val Gln 355 360 365 ctg aat tcc tgc ctg gct gct ggt gacatc cta gcc ctg gtt ttt ggc 1152 Leu Asn Ser Cys Leu Ala Ala Gly Asp IleLeu Ala Leu Val Phe Gly 370 375 380 ctc ctt ttt gct gtc acc agc gtc gcgttc ctt gtg cag atg aga agg 1200 Leu Leu Phe Ala Val Thr Ser Val Ala PheLeu Val Gln Met Arg Arg 385 390 395 400 cag cac aga agg gga acc aaa gggggt gtg agc tac cgc cca gca gag 1248 Gln His Arg Arg Gly Thr Lys Gly GlyVal Ser Tyr Arg Pro Ala Glu 405 410 415 gta gcc gag act gga gcctagaggctgg atcttggaga atgtgagaag 1296 Val Ala Glu Thr Gly Ala 420ccagccagag gcatctgagg gggagccggt aactgtcctg tcctgctcat tatgccactt 1356ccttttaact gccaagaaat tttttaaaat aaatatttat aat 1399 2 422 PRT HUMAN 2Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro Leu Gly Gly Gly Ser 1 5 1015 Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp Leu Pro Ser Glu Glu 20 2530 Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu Glu Asp Leu Pro Gly 35 4045 Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Glu Val Lys Pro Lys 50 5560 Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp Leu Pro Thr Val Glu 65 7075 80 Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn Ala His Arg Asp Lys 8590 95 Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro Pro Trp100 105 110 Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe Gln Ser Pro ValAsp 115 120 125 Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala Leu Arg ProLeu Glu 130 135 140 Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu Leu ArgLeu Arg Asn 145 150 155 160 Asn Gly His Ser Val Gln Leu Thr Leu Pro ProGly Leu Glu Met Ala 165 170 175 Leu Gly Pro Gly Arg Glu Tyr Arg Ala LeuGln Leu His Leu His Trp 180 185 190 Gly Ala Ala Gly Arg Pro Gly Ser GluHis Thr Val Glu Gly His Arg 195 200 205 Phe Pro Ala Glu Ile His Val ValHis Leu Ser Thr Ala Phe Ala Arg 210 215 220 Val Asp Glu Ala Leu Gly ArgPro Gly Gly Leu Ala Val Leu Ala Ala 225 230 235 240 Phe Leu Glu Glu GlyPro Glu Glu Asn Ser Ala Tyr Glu Gln Leu Leu 245 250 255 Ser Arg Leu GluGlu Ile Ala Glu Glu Gly Ser Glu Thr Gln Val Pro 260 265 270 Gly Leu AspIle Ser Ala Leu Leu Pro Ser Asp Phe Ser Arg Tyr Phe 275 280 285 Gln TyrGlu Gly Ser Leu Thr Thr Pro Pro Cys Ala Gln Gly Val Ile 290 295 300 TrpThr Val Phe Asn Gln Thr Val Met Leu Ser Ala Lys Gln Leu His 305 310 315320 Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp Ser Arg Leu Gln Leu 325330 335 Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg Val Ile Glu Ala Ser340 345 350 Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala Ala Glu Pro ValGln 355 360 365 Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu Ala Leu ValPhe Gly 370 375 380 Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu Val GlnMet Arg Arg 385 390 395 400 Gln His Arg Arg Gly Thr Lys Gly Gly Val SerTyr Arg Pro Ala Glu 405 410 415 Val Ala Glu Thr Gly Ala 420 3 29 DNAHUMAN 3 cgcccagtgg gtcatcttcc ccagaagag 29 4 19 DNA HUMAN 4 ggaatcctcctgcatccgg 19 5 1522 DNA HUMAN CDS (13)..(1389) mat_peptide (124)..(1389)5 acagtcagcc gc atg gct ccc ctg tgc ccc agc ccc tgg ctc cct ctg ttg 51Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu -35 -30 -25 atc ccggcc cct gct cca ggc ctc act gtg caa ctg ctg ctg tca ctg 99 Ile Pro AlaPro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu -20 -15 -10 ctg cttctg atg cct gtc cat ccc cag agg ttg ccc cgg atg cag gag 147 Leu Leu LeuMet Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu -5 -1 1 5 gat tccccc ttg gga gga ggc tct tct ggg gaa gat gac cca ctg ggc 195 Asp Ser ProLeu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu Gly 10 15 20 gag gag gatctg ccc agt gaa gag gat tca ccc aga gag gag gat cca 243 Glu Glu Asp LeuPro Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro 25 30 35 40 ccc gga gaggag gat cta cct gga gag gag gat cta cct gga gag gag 291 Pro Gly Glu GluAsp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu 45 50 55 gat cta cct gaagtt aag cct aaa tca gaa gaa gag ggc tcc ctg aag 339 Asp Leu Pro Glu ValLys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys 60 65 70 tta gag gat cta cctact gtt gag gct cct gga gat cct caa gaa ccc 387 Leu Glu Asp Leu Pro ThrVal Glu Ala Pro Gly Asp Pro Gln Glu Pro 75 80 85 cag aat aat gcc cac agggac aaa gaa ggg gat gac cag agt cat tgg 435 Gln Asn Asn Ala His Arg AspLys Glu Gly Asp Asp Gln Ser His Trp 90 95 100 cgc tat gga ggc gac ccgccc tgg ccc cgg gtg tcc cca gcc tgc gcg 483 Arg Tyr Gly Gly Asp Pro ProTrp Pro Arg Val Ser Pro Ala Cys Ala 105 110 115 120 ggc cgc ttc cag tccccg gtg gat atc cgc ccc cag ctc gcc gcc ttc 531 Gly Arg Phe Gln Ser ProVal Asp Ile Arg Pro Gln Leu Ala Ala Phe 125 130 135 tgc ccg gcc ctg cgcccc ctg gaa ctc ctg ggc ttc cag ctc ccg ccg 579 Cys Pro Ala Leu Arg ProLeu Glu Leu Leu Gly Phe Gln Leu Pro Pro 140 145 150 ctc cca gaa ctg cgcctg cgc aac aat ggc cac agt gtg caa ctg acc 627 Leu Pro Glu Leu Arg LeuArg Asn Asn Gly His Ser Val Gln Leu Thr 155 160 165 ctg cct cct ggg ctagag atg gct ctg ggt ccc ggg cgg gag tac cgg 675 Leu Pro Pro Gly Leu GluMet Ala Leu Gly Pro Gly Arg Glu Tyr Arg 170 175 180 gct ctg cag ctg catctg cac tgg ggg gct gca ggt cgt ccg ggc tcg 723 Ala Leu Gln Leu His LeuHis Trp Gly Ala Ala Gly Arg Pro Gly Ser 185 190 195 200 gag cac act gtggaa ggc cac cgt ttc cct gcc gag atc cac gtg gtt 771 Glu His Thr Val GluGly His Arg Phe Pro Ala Glu Ile His Val Val 205 210 215 cac ctc agc accgcc ttt gcc aga gtt gac gag gcc ttg ggg cgc ccg 819 His Leu Ser Thr AlaPhe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro 220 225 230 gga ggc ctg gccgtg ttg gcc gcc ttt ctg gag gag ggc ccg gaa gaa 867 Gly Gly Leu Ala ValLeu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu 235 240 245 aac agt gcc tatgag cag ttg ctg tct cgc ttg gaa gaa atc gct gag 915 Asn Ser Ala Tyr GluGln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu 250 255 260 gaa ggc tca gagact cag gtc cca gga ctg gac ata tct gca ctc ctg 963 Glu Gly Ser Glu ThrGln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu 265 270 275 280 ccc tct gacttc agc cgc tac ttc caa tat gag ggg tct ctg act aca 1011 Pro Ser Asp PheSer Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr 285 290 295 ccg ccc tgtgcc cag ggt gtc atc tgg act gtg ttt aac cag aca gtg 1059 Pro Pro Cys AlaGln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val 300 305 310 atg ctg agtgct aag cag ctc cac acc ctc tct gac acc ctg tgg gga 1107 Met Leu Ser AlaLys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly 315 320 325 cct ggt gactct cgg cta cag ctg aac ttc cga gcg acg cag cct ttg 1155 Pro Gly Asp SerArg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu 330 335 340 aat ggg cgagtg att gag gcc tcc ttc cct gct gga gtg gac agc agt 1203 Asn Gly Arg ValIle Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser 345 350 355 360 cct cgggct gct gag cca gtc cag ctg aat tcc tgc ctg gct gct ggt 1251 Pro Arg AlaAla Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly 365 370 375 gac atccta gcc ctg gtt ttt ggc ctc ctt ttt gct gtc acc agc gtc 1299 Asp Ile LeuAla Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val 380 385 390 gcg ttcctt gtg cag atg aga agg cag cac aga agg gga acc aaa ggg 1347 Ala Phe LeuVal Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly 395 400 405 ggt gtgagc tac cgc cca gca gag gta gcc gag act gga gcc 1389 Gly Val Ser Tyr ArgPro Ala Glu Val Ala Glu Thr Gly Ala 410 415 420 tagaggctgg atcttggagaatgtgagaag ccagccagag gcatctgagg gggagccggt 1449 aactgtcctg tcctgctcattatgccactt ccttttaact gccaagaaat tttttaaaat 1509 aaatatttat aat 1522 6459 PRT HUMAN 6 Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu IlePro Ala -35 -30 -25 Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser LeuLeu Leu Leu -20 -15 -10 Met Pro Val His Pro Gln Arg Leu Pro Arg Met GlnGlu Asp Ser Pro -5 -1 1 5 10 Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp ProLeu Gly Glu Glu Asp 15 20 25 Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu GluAsp Pro Pro Gly Glu 30 35 40 Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro GlyGlu Glu Asp Leu Pro 45 50 55 Glu Val Lys Pro Lys Ser Glu Glu Glu Gly SerLeu Lys Leu Glu Asp 60 65 70 75 Leu Pro Thr Val Glu Ala Pro Gly Asp ProGln Glu Pro Gln Asn Asn 80 85 90 Ala His Arg Asp Lys Glu Gly Asp Asp GlnSer His Trp Arg Tyr Gly 95 100 105 Gly Asp Pro Pro Trp Pro Arg Val SerPro Ala Cys Ala Gly Arg Phe 110 115 120 Gln Ser Pro Val Asp Ile Arg ProGln Leu Ala Ala Phe Cys Pro Ala 125 130 135 Leu Arg Pro Leu Glu Leu LeuGly Phe Gln Leu Pro Pro Leu Pro Glu 140 145 150 155 Leu Arg Leu Arg AsnAsn Gly His Ser Val Gln Leu Thr Leu Pro Pro 160 165 170 Gly Leu Glu MetAla Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln 175 180 185 Leu His LeuHis Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr 190 195 200 Val GluGly His Arg Phe Pro Ala Glu Ile His Val Val His Leu Ser 205 210 215 ThrAla Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu 220 225 230235 Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala 240245 250 Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser255 260 265 Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro SerAsp 270 275 280 Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr ProPro Cys 285 290 295 Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr ValMet Leu Ser 300 305 310 315 Ala Lys Gln Leu His Thr Leu Ser Asp Thr LeuTrp Gly Pro Gly Asp 320 325 330 Ser Arg Leu Gln Leu Asn Phe Arg Ala ThrGln Pro Leu Asn Gly Arg 335 340 345 Val Ile Glu Ala Ser Phe Pro Ala GlyVal Asp Ser Ser Pro Arg Ala 350 355 360 Ala Glu Pro Val Gln Leu Asn SerCys Leu Ala Ala Gly Asp Ile Leu 365 370 375 Ala Leu Val Phe Gly Leu LeuPhe Ala Val Thr Ser Val Ala Phe Leu 380 385 390 395 Val Gln Met Arg ArgGln His Arg Arg Gly Thr Lys Gly Gly Val Ser 400 405 410 Tyr Arg Pro AlaGlu Val Ala Glu Thr Gly Ala 415 420 7 25 DNA HUMAN 7 tggggttcttgaggatctcc aggag 25 8 26 DNA HUMAN 8 ctctaacttc agggagccct cttctt 26 948 DNA HUMAN primer_bind (1)..(48) 9 cuacuacuac uaggccacgc gtcgactagtacgggnnggg nngggnng 48 10 6 PRT HUMAN 10 Glu Glu Asp Leu Pro Ser 1 5 116 PRT HUMAN 11 Gly Glu Asp Asp Pro Leu 1 5 12 21 PRT HUMAN 12 Asn AsnAla His Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp Arg 1 5 10 15 TyrGly Gly Asp Pro 20 13 16 PRT HUMAN 13 His Pro Gln Arg Leu Pro Arg MetGln Glu Asp Ser Pro Leu Gly Gly 1 5 10 15 14 24 PRT HUMAN 14 Glu Glu AspSer Pro Arg Glu Glu Asp Pro Pro Gly Glu Glu Asp Leu 1 5 10 15 Pro GlyGlu Glu Asp Leu Pro Gly 20 15 13 PRT HUMAN 15 Leu Glu Glu Gly Pro GluGlu Asn Ser Ala Tyr Glu Gln 1 5 10 16 16 PRT HUMAN 16 Met Arg Arg GlnHis Arg Arg Gly Thr Lys Gly Gly Val Ser Tyr Arg 1 5 10 15 17 45 DNAHUMAN 17 gtcgctagct ccatgggtca tatgcagagg ttgccccgga tgcag 45 18 43 DNAHUMAN 18 gaagatctct tactcgagca ttctccaaga tccagcctct agg 43 19 10 DNAHUMAN misc_feature (1)..(10) 19 yssccmnsss 10 20 10 DNA HUMANmisc_feature (1)..(10) 20 kmggcckrry 10 21 10 DNA HUMAN misc_feature(1)..(10) 21 rrrcwwgyyy 10 22 8 PRT HUMAN 22 Leu Glu His His His His HisHis 1 5 23 5052 DNA HUMAN misc_feature (1)..(5052) 23 ggatcctgttgactcgtgac cttaccccca accctgtgct ctctgaaaca tgagctgtgt 60 ccactcagggttaaatggat taagggcggt gcaagatgtg ctttgttaaa cagatgcttg 120 aaggcagcatgctcgttaag agtcatcacc aatccctaat ctcaagtaat cagggacaca 180 aacactgcggaaggccgcag ggtcctctgc ctaggaaaac cagagacctt tgttcacttg 240 tttatctgaccttccctcca ctattgtcca tgaccctgcc aaatccccct ctgtgagaaa 300 cacccaagaattatcaataa aaaaataaat ttaaaaaaaa aatacaaaaa aaaaaaaaaa 360 aaaaaaaaaagacttacgaa tagttattga taaatgaata gctattggta aagccaagta 420 aatgatcatattcaaaacca gacggccatc atcacagctc aagtctacct gatttgatct 480 ctttatcattgtcattcttt ggattcacta gattagtcat catcctcaaa attctccccc 540 aagttctaattacgttccaa acatttaggg gttacatgaa gcttgaacct actaccttct 600 ttgcttttgagccatgagtt gtaggaatga tgagtttaca ccttacatgc tggggattaa 660 tttaaactttacctctaagt cagttgggta gcctttggct tatttttgta gctaattttg 720 tagttaatggatgcactgtg aatcttgcta tgatagtttt cctccacact ttgccactag 780 gggtaggtaggtactcagtt ttcagtaatt gcttacctaa gaccctaagc cctatttctc 840 ttgtactggcctttatctgt aatatgggca tatttaatac aatataattt ttggagtttt 900 tttgtttgtttgtttgtttg tttttttgag acggagtctt gcatctgtca tgcccaggct 960 ggagtagcagtggtgccatc tcggctcact gcaagctcca cctcccgagt tcacgccatt 1020 ttcctgcctcagcctcccga gtagctggga ctacaggcgc ccgccaccat gcccggctaa 1080 ttttttgtatttttggtaga gacggggttt caccgtgtta gccagaatgg tctcgatctc 1140 ctgacttcgtgatccacccg cctcggcctc ccaaagttct gggattacag gtgtgagcca 1200 ccgcacctggccaatttttt gagtctttta aagtaaaaat atgtcttgta agctggtaac 1260 tatggtacatttccttttat taatgtggtg ctgacggtca tataggttct tttgagtttg 1320 gcatgcatatgctacttttt gcagtccttt cattacattt ttctctcttc atttgaagag 1380 catgttatatcttttagctt cacttggctt aaaaggttct ctcattagcc taacacagtg 1440 tcattgttggtaccacttgg atcataagtg gaaaaacagt caagaaattg cacagtaata 1500 cttgtttgtaagagggatga ttcaggtgaa tctgacacta agaaactccc ctacctgagg 1560 tctgagattcctctgacatt gctgtatata ggcttttcct ttgacagcct gtgactgcgg 1620 actatttttcttaagcaaga tatgctaaag ttttgtgagc ctttttccag agagaggtct 1680 catatctgcatcaagtgaga acatataatg tctgcatgtt tccatatttc aggaatgttt 1740 gcttgtgttttatgctttta tatagacagg gaaacttgtt cctcagtgac ccaaaagagg 1800 tgggaattgttattggatat catcattggc ccacgctttc tgaccttgga aacaattaag 1860 ggttcataatctcaattctg tcagaattgg tacaagaaat agctgctatg tttcttgaca 1920 ttccacttggtaggaaataa gaatgtgaaa ctcttcagtt ggtgtgtgtc cctngttttt 1980 ttgcaatttccttcttactg tgttaaaaaa aagtatgatc ttgctctgag aggtgaggca 2040 ttcttaatcatgatctttaa agatcaataa tataatcctt tcaaggatta tgtctttatt 2100 ataataaagataatttgtct ttaacagaat caataatata atcccttaaa ggattatatc 2160 tttgctgggcgcagtggctc acacctgtaa tcccagcact ttgggtggcc aaggtggaag 2220 gatcaaatttgcctacttct atattatctt ctaaagcaga attcatctct cttccctcaa 2280 tatgatgatattgacagggt ttgccctcac tcactagatt gtgagctcct gctcagggca 2340 ggtagngttttttgtttttg tttttgtttt tcttttttga gacagggtct tgctctgtca 2400 cccaggccagagtgcaatgg tacagtctca gctcactgca gcctcaacgc ctcggctcaa 2460 accatcatcccatttcagcc tcctgagtag ctgggactac aggcacatgc cattacacct 2520 ggctaatttttttgtatttc tagtagagac agggtttggc catgttgccc gggctggtct 2580 cgaactcctggactcaagca atccacccac ctcagcctcc caaaatgagg gaccgtgtct 2640 tattcatttccatgtcccta gtccatagcc cagtgctgga cctatggtag tactaaataa 2700 atatttgttgaatgcaatag taaatagcat ttcagggagc aagaactaga ttaacaaagg 2760 tggtaaaaggtttggagaaa aaaataatag tttaatttgg ctagagtatg agggagagta 2820 gtaggagacaagatggaaag gtctcttggg caaggttttg aaggaagttg gaagtcagaa 2880 gtacacaatgtgatatcgtg gcaggcagtg gggagccaat gaaggctttt gagcaggaga 2940 gtaatgtgttgaaaaataaa tataggttaa acctatcaga gcccctctga cacatacact 3000 tgcttttcattcaagctcaa gtttgtctcc cacataccca ttacttaact caccctcggg 3060 ctcccctagcagcctgccct acctctttac ctgcttcctg gtggagtcag ggatgtatac 3120 atgagctgctttccctctca gccagagaca tggggggccc cagctcccct gcctttcccc 3180 ttctgtgcctggagctggga agcaggccag ggttagctga ggctggctgg caagcagctg 3240 ggtggtgccagggagagcct gcatagtgcc aggtggtgcc ttgggttcca agctagtcca 3300 tggccccgataaccttctgc ctgtgcacac acctgcccct cactccaccc ccatcctagc 3360 tttggtatgggggagagggc acagggccag acaaacctgt gagactttgg ctccatctct 3420 gcaaaagggcgctctgtgag tcagcctgct cccctccagg cttgctcctc ccccacccag 3480 ctctcgtttccaatgcacgt acagcccgta cacaccgtgt gctgggacac cccacagtca 3540 gcgcatggctcccctgtgcc ccagcccctg gctccctctg ttgatcccgg cccctgctcc 3600 aggcctcactgtgcaactgc tgctgtcact gctgcttctg atgcctgtcc atccccagag 3660 gttgccccggatgcaggagg attccccctt ggaggaggct cttctgggga agatgaccca 3720 ctgggcgaggaggatctgcc cagtgaagag gattcaccca gagaggagga tccacccgga 3780 gaggaggatctacctggaga ggaggatcta cctggagagg aggatctacc tgaagttaat 3840 gcctaaatcagaagaagagg gctccctgaa gttagaggat ctacctactg ttgaggctcc 3900 tggagatcctcaagaacccc agaataatgc ccacagggac aaagaagggg atgaccagag 3960 tcattggcgctatggaggcg acccgcctgg ccccgggtgt ccccagcctg cgcgggccgc 4020 ttccagtccccggtggatat ccgcccccag ctcgccgcct tctgcccggc cctgcgcccc 4080 ctggaactcctgggcttcca gctcccgccg ctcccagaac tgcgcctgca gacaatggcc 4140 acagtgtgcaactgaccctg cctcctgggc tagagatggc tctgggtccc gggcgggagt 4200 accggctctgcagctgcatc tgcactgggg ggctgcaggt cgtccgggct cggagcacac 4260 tgtggaaggccaccgtttcc ctgccgagat ccacgtggtt cacctcagca ccgcctttgc 4320 cagagttgacgaggccttgg ggcgcccggg aggcctggcc gtgttggcgc ctttctggag 4380 gagggcccggaagaaaacag tgtcctatga gcagttgctg tctcgcttgg aagaaatcgc 4440 tgaggaaggctcagagactc aggtcccagg actggacata tctgcactcc tgccctctga 4500 cttcagccgctacttccaat atgaggggtc tctgactaca ccgccctgtg cccagggtgt 4560 catctggactgtgtttaacc agacagtgat gctgagtgct aagcagctcc acaccctctc 4620 tgacaccctgtggggacctg gtgactctcg gctacagctg aacttccgag cgacgcagcc 4680 tttgaatgggcgagtgattg aggcctcctt ccctgctgga gtggacagca gtcctcgggc 4740 tgctgagccagtccagctga attcctgcct ggctgctggt gacatcctag ccctggtttt 4800 tggcctcctttttgctgtca ccagcgtcgc gttccttgtg cagatgagaa ggcagcacag 4860 aaggggaaccaaagggggtg tgagcgtacc gcccagcaga ggtagccgag actggagcct 4920 agaggctggatcttggagaa tgtgagaagc cagccagagg catctgaggg ggagccggta 4980 actgtcctgtcctgctcatt atgccacttc cttttaactg ccaagaaatt ttttaaaata 5040 aatatttataat 5052 24 6 PRT HUMAN 24 Arg Arg Ala Arg Lys Lys 1 5 25 4 PRT HUMANSITE (1)..(4) 25 Ser Pro Xaa Xaa 1 26 4 PRT HUMAN SITE (1)..(4) 26 ThrPro Xaa Xaa 1

1-30 (canceled).
 31. An MN antisense construct comprising an MNantisense oligonucleotide operably linked to an expression controlsequence in a vector, wherein said MN antisense oligonucleotide iscomplementary to SEQ ID NO: 5, and wherein said MN antisense constructshows antisense activity in an in vitro screening assay comprising thesteps of: (a) contacting a human cell abnormally expressing MN with saidMN antisense construct; (b) determining the effect of said MN antisenseconstruct on MN expression in said human cell; and (c) concluding thatif MN expression is decreased, that said MN antisense construct showsantisense activity.
 32. The MN antisense construct of claim 31, whereinsaid MN antisense nucleotide is complementary to the 5′ end of the mRNAthat is transcribed from the complement of SEQ ID NO:
 5. 33. The MNantisense construct of claim 31, wherein said MN antisense nucleotide iscomplementary to SEQ ID NO:
 1. 34. The MN antisense construct of claim31, wherein said vector is derived from a plasmid, a cosmid, abacteriophage or a virus.
 35. A composition comprising apharmaceutically acceptable carrier and the MN antisense construct ofclaim 31, wherein said MN antisense construct interacts with MN gene orMN transcript.
 36. An MN antisense construct comprising an MN antisensedouble-stranded ribonucleic acid operably linked to an expressioncontrol sequence in a vector, wherein said MN antisense double-strandedribonucleic acid consists of a nucleotide sequence that is complementaryto SEQ ID NO: 5, and wherein said MN antisense double-strandedribonucleic acid shows antisense activity in an in vitro screening assaycomprising the steps of: (a) contacting a human cell abnormallyexpressing MN with said MN antisense double-stranded ribonucleic acid;(b) determining the effect of said MN antisense double-strandedribonucleic acid on MN expression in said human cell; and (c) concludingthat if MN expression is decreased, that said MN antisensedouble-stranded ribonucleic acid shows antisense activity.
 37. The MNantisense construct of claim 36, wherein said vector is derived from aplasmid, a cosmid, a bacteriophage or a virus.
 38. A compositioncomprising a pharmaceutically acceptable carrier and the MN antisenseconstruct according to claim 36, wherein said MN antisense constructinteracts with MN gene or MN transcript.
 39. A method of blocking invivo expression of the MN gene in a human by administering an MNantisense construct of claim
 31. 40. A method of treating neoplasticdisease and/or pre-neoplastic disease in a human, wherein said diseaseis associated with abnormal MN gene expression, comprising inhibitingthe expression of MN gene by administering an MN antisense constructaccording to claim
 31. 41. An antibody which specifically binds to an MNprotein or to an MN polypeptide, wherein said MN protein or MNpolypeptide is encoded by a nucleic acid that comprises a polynucleotidecontaining at least 29 nucleotides, said nucleic acid being selectedfrom the group consisting of: (a) SEQ ID NO: 5; (b) polynucleotides thathybridize under stringent conditions to SEQ ID NO: 5's complement; and(c) polynucleotides that differ from SEQ ID NO: 5 or from thepolynucleotide sequences of (b) due to the degeneracy of the geneticcode, and wherein said antibody is polyclonal.
 42. The antibody of claim41 that is conjugated to a toxin.
 43. The antibody of claim 41 that isconjugated to a chemotherapeutic drug.
 44. An antibody whichspecifically binds to an MN protein or to an MN polypeptide, whereinsaid MN protein or MN polypeptide is encoded by a nucleic acid thatcomprises a polynucleotide containing at least 29 nucleotides, saidnucleic acid being selected from the group consisting of: (a) SEQ ID NO:5; (b) polynucleotides that hybridize under stringent conditions to SEQID NO: 5's complement; and (c) polynucleotides that differ from SEQ IDNO: 5 or from the polynucleotide sequences of (b) due to the degeneracyof the genetic code, and wherein said antibody is humanized.
 45. Theantibody of claim 44 that is conjugated to a toxin.
 46. The antibody ofclaim 44 that is conjugated to a chemotherapeutic drug.